CN102908631B - The function of monomethylation of actin 84 lysine in cytokinesis and cell proliferation and its application in drug development - Google Patents

Abstract

The present invention discloses a new methylation modification of actin, that is, the function of actin K84me1 modified by monomethylation of lysine 84 in cytokinesis and cell proliferation and its potential in the research and development of anticancer drugs targeting actin K84me1 Value. NMII can slide along actin fibers only after ALKBH4 protein demethylates actinK84me1. ActinK84me1 inhibits contractile ring contraction and interferes with cytokinesis. ALKBH4 gene silencing can lead to increased expression of actin K84me1, and overexpression of actin K84me1 leads to cell division arrest and cytokinesis failure, eventually causing cell apoptosis and multinucleated cell generation, resulting in defects in cell proliferation and migration and eventually death. The expression level of actin K84me1 can be used in the development of antitumor drugs.

Description

84 lysine monomethylations of actin are modified at the function in cytokinesis and cell proliferation and apply in medicament research and development

Technical field

The present invention relates to 84 lysine monomethylations modification (actin K84me1) functions in cytokinesis and cell proliferation of actin reaches for the potential using value in actin K84me1 cancer drug development.

Background technology

Cytokinesis is four main stages of experience usually: split plate is established (cleavage plane specification), minute dehiscence furrow contraction (cleavage furrow ingression), middle body forms (midbody formation) and fracture (abscission).The cytokinesis failure can cause centrosome over-replicate genomic instability usually, and these all can cause pernicious increment and migration.In order to guarantee that on room and time cytokinesis correctly occurs, many components of cytokinesis and other cell pathway process all participate in this regulation process closely.The retraction ring (contractile ring) that cytokinesis is considered to form by actin fiber (F-actin) and the non-muscle myosin of two types (non-muscle myosin II or NM II) has usually shunk, and the actin fiber plays key effect in minute dehiscence furrow formation and contraction process.Retraction ring is the height dynamic change along with the fast transition between actin fiber and the non-muscle myosin of two types, and the actin transmitting fiber tow that the actin fiber is the one-tenth nuclear effect by dividing dehiscence furrow place actin fiber or the nucleation that other positions have existed in minute dehiscence furrow place gathering is delivered to minute dehiscence furrow place.The non-muscle myosin of two types is gathered in a minute dehiscence furrow place and does not rely on the atpase activity of himself, but depends on the phosphorylation modification of light chain.The non-muscle myosin of two types is dynein main in the cytokinesis process, and it slides and promote that the depolymerization of actin fiber is all that minute dehiscence furrow contraction is necessary along the actin fiber.Actin and the myosin height change at minute dehiscence furrow place is to be subject to protein post-translational modification institute (post-translational modifcations or PTMs) regulation and control, there is the research table to show that recruiting a minute dehiscence furrow place at mitotic early anaphase myosin completes by phosphorylation modification, promote the kinases degraded of phosphorylation can cause myosin location and actin in a minute dehiscence furrow place depolymerization, the depolymerization of actin and polymerization also are subject to the signal path regulation and control of multiple post translational modification, for example oxidation, arginyl, SUMOization and S-nitrosoglutathione, these are to the actin structure, distribution and function have material impact, yet whether the actin on retraction ring and middle body has similar modification, these modifications are that how to affect the 26S Proteasome Structure and Function of body in retraction ring now not clear.

Cytokinesis is an astonishing complicated regulation process, needs very many interaction component and regulatory pathway, and the failure of cytokinesis can result from the result of certain component inactivation in any one-phase of this four-stage or excessive activation.Although the albumen of many cytokinesiss is identified, we just know how interactional these albumen are and how to be regulated and controled.It is unsuccessfully very important understanding cytokinesis, because it may be the reason that produces unsettled tetraploid cell and tumor.Yet what cytokinesis failure also can physiological regulation occurs in some tissues, even those do not have the tissue of tumor tendency, as heart.How to understand in the situation that reply unlike signal or be under pressure and damage physiological regulation and control cytokinesis at cell, be still the field of following primary study.Although cytokinesis unsuccessfully is accompanied by some pathological phenomenons, as cancer, drug-induced cytokinesis unsuccessfully can be used as the important directions of new drug development.The Rho inhibitors of kinases has been developed into cardiovascular drugs, in the middle of the Aurora A inhibitor being is is being researched and developed as cancer therapy drug.Because these inhibitor also can be induced normal structure cytokinesis failure, so determine that different tissues is very important to the consequence of cytokinesis sensitivity and different tissues generation tetraploid cell.

Summary of the invention

The invention discloses the modification that methylates that a kind of actin is new, 84 lysine monomethylations modification (actin K84me1) functions in cytokinesis and cell proliferation reach for the potential using value in actin K84me1 cancer drug development.Described people ALKBH4 cDNA sequence is as shown in sequence table SEQ ID NO.1, described people ALKBH4 H169A-D171A-H254A mutants cDNA sequence is as sequence table SEQ IDNO.2, shown in described mice ALKBH4 genomic dna sequence as shown in sequence table SEQ ID NO.3, described people β-actin cDNA sequence is as shown in sequence table SEQ ID NO.4, described people β-actin K84A mutants cDNA sequence is as shown in sequence table SEQ ID NO.5, described people ALKBH4 protein sequence is as shown in sequence table SEQ ID NO.6, described people ALKBH4 H169A-D171A-H254A mutant protein sequence is as shown in sequence table SEQ ID NO.7, described mice ALKBH4 protein sequence is as shown in sequence table SEQ ID NO.8, described people β-actin protein sequence is as shown in sequence table SEQ ID NO.9, described people β-actin K84A mutant protein sequence is as shown in sequence table SEQ ID NO.10.Described ALKBH4 siRNA sequence SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, effect is by the ALKBH4 gene silencing, degraded ALKBH4 cDNA and the endogenous ALKBH4 protein expression of reduction, the target spot of these three sequences is positioned at 3 ' the end noncoding region of ALKBH4 mRNA, so can not reticent heterogenous expression ALKBH4 gene.Described NM II siRNA sequence SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32 effect are by NM II gene silencing, degraded NM II cDNA and reduction NM II protein expression.

At first, immunofluorescence and immunoblot experiment detect actin (actin) and ALKBH4 protein localization on retraction ring (contractile ring) and middle body (midbody), mass-spectrometric technique finds that 84 lysines of actin have monomethylation and modify (actin K84me1), immunofluorescence technique detects retraction ring and the middle body that actin K84me1 also is positioned at cell, so technical solution problem one of the present invention provides a kind of new modified forms actin K84me1 of actin albumen and its organelle specific localization phenotype.

Secondly, co-immunoprecipitation and mass-spectrometric technique find that ALKBH4 albumen and actin (actin) and the non-muscle myosin of two types (NM II) interact, ALKBH4 albumen and the specific interaction of actin K84me1, and NM II interacts with the actin modified that do not methylate, and actin K84me1 disturbs NM II combination, suppress NM II fiber and slide along the actin fiber, this depression effect interferes with retraction ring and shrinks, and destroys the cytokinesis process.Only at ALKBH4 albumen, NM II after actin K84me1 demethylation could be slided along the actin fiber.Therefore technical solution problem two of the present invention provides actin K84me1 disturb NM II combination and suppress NM II and slide along the actin fiber, and actinK84me1 inhibition retraction ring shrinks and disturbs cytokinesis.

Finally, the present invention finds that by gene silencing, immunofluorescence and Flow Cytometry the ALKBH4 gene silencing can cause actin K84me1 expression to increase, actin K84me1 crosses to express and causes cell division phase retardance and cytokinesis failure, finally cause that apoptosis and apocyte generate, actin K84me1 crosses expression can cause cell proliferation migration defect finally deathward.So technical solution problem three of the present invention provides the basic research evidence for its death of tumor cell induction that suppresses the malignant proliferation migration, this just provides strong theoretical basis for the antitumor drug research and development that our research is crossed expression for actin K84me1.

As everyone knows, malignant cell is can wireless propagation and the cell of migration, and Normocellular vivosphere has been occupied in its pernicious amplification, causes that numerous organ failuries seize patients ' lives.And we find that actin K84me1 plays crucial regulating and controlling effect in especially cytokinesis process of cell division, if actin K84me1 expression in the increase tumor cell, tumor cell can't breed and move and finally cause its death so, thereby reach the purpose for the treatment of malignant tumor.In sum, illustrating can be for researching and developing antitumor drug for actin K84me1 expression.

The accompanying drawing explanation

Fig. 1 human dioxygenase AlkB protein family forms and enzymatic activity key structure territory

Fig. 2 dioxygenase AlkB protein family has identical demethylation mechanism with histone demethylase JHDM protein family

Fig. 3 affinitive layer purification 6 * His-ALKBH4 wild type and HDH mutant fusion protein

Fig. 4 concentrates 6 * His-ALKBH4 wild type and HDH mutant fusion protein and verifies the purity of these two albumen

Fig. 5 verifies GST-ALKBH4 fusion rotein solubility expression, affinity chromatograph and ion-exchange chromatography separation and purification GST-ALKBH4 fusion rotein

Fig. 6 affinitive layer purification is for the rabbit source ALKBH4 antibody of immunofluorescence experiment

The specificity of Fig. 7 immunofluorescence experiment checking rabbit source ALKBH4 antibody

Fig. 8 is for the preparation of the antibody of immunoblot experiment and the specificity of immunoblot experiment checking antibody

Fig. 9 immunofluorescence experiment proves that endogenous ALKBH4 protein localization is on centrosome (centrosome) and middle body (midbody)

Figure 10 immunofluorescence experiment proof external source 3 * Flag-ALKBH4 is positioned on centrosome, and the centrosome separating experiment proves that endogenous ALKBH4 is positioned on centrosome

In Figure 11 immunofluorescence experiment and retraction ring, body separating experiment proof ALKBH4 is positioned on retraction ring (contractile ring) and middle body

Figure 12 ALKBH4 co-immunoprecipitation mass spectrometry results, actin K84me1 and NM II are present in ALKBH4 co-immunoprecipitation sample

Figure 13 co-immunoprecipitation experimental results show that endogenous and ALKBH4 external source interacts with actin

Figure 14 co-immunoprecipitation experimental results show that HA-β-actin K84A mutant disturbs the ALKBH4 combination, and the binding ability of Flag-GFP-ALKBH4 HDH mutant and actin strengthens

Figure 15 co-immunoprecipitation experimental results show that ALKBH4 and NM II interact, and HA-β-actin K84A mutant disturbs the combination between itself and NM II

Figure 16 co-immunoprecipitation experimental results show that the ALKBH4 gene silencing does not affect the interaction between actin and NM II

Figure 17 co-immunoprecipitation experimental results show that NM II gene silencing does not affect ALKBH4 and actin interphase interaction

Figure 18 co-immunoprecipitation experimental results show that NM II inhibitor B lebbistatin does not affect ALKBH4 and actin interphase interaction

Figure 19 co-immunoprecipitation experimental results show that actin polymerization inhibitor Cytochalasin D has destroyed the interaction between ALKBH4 and actin, NM II

Polypeptide and HA-β-actin that Figure 20 rabbit source actin K84me1 antibody can specific recognition contains K84me1, can not identify non-methylated polypeptide and HA-β-actin K84A mutant

Figure 21 immunofluorescence experiment checking actin K84me1 is positioned on retraction ring and middle body

Figure 22 ALKBH4 in vivo can make actin K84me1 demethylation, and ALKBH4 is combined with actin K84me1 and NM II is combined with the actin modified that do not methylate

Figure 23 immunofluorescence experiment proof ALKBH4 gene silencing causes the retraction ring malposition, middle body structure is abnormal and the centrosome over-replicate

Figure 24 Flow Cytometry proof ALKBH4 gene silencing causes G2/M phase and M phase cytosis

Figure 25 crosses expression 3 * Flag-ALKBH4 HDH and HA-β-actin K84A mutant causes M phase cytosis

Figure 26 immunofluorescence, Flow Cytometry and living cells imaging experiment proof ALKBH4 gene silencing cause final apocyte, the apoptosis of producing of cytokinesis failure

Figure 27 Flag-GFP-ALKBH4 wild type can be repaired the cytokinesis phenomenon that the ALKBH4 gene silencing causes, and comprises that the retraction ring location is abnormal, and apocyte produces and M phase cytosis, and Flag-GFP-ALKBH4 HDH mutant can not

Figure 28 ALKBH4 is at intracellular functional mode

In Figure 29 Flow Cytometry proof U2OS cell, the ALKBH4 gene silencing causes G2/M phase, M phase cytosis and apoptosis

In Figure 30 Flow Cytometry proof OSC20 and TOC2 cell, the ALKBH4 gene silencing causes apoptosis

The albumen position that Figure 31 ALKBH4 knock out mice knocks out and traditional ALKBH4 knock out mice are died from the period of embryo

Figure 32 condition type ALKBH4 knock out mice design and southern blotting and RT-PCR checking

Figure 33 induces ALKBH4 knock out mice fibroblast ALKBH4 gene knockout to cause that actin K84me1 expression increases

Figure 34 induces ALKBH4 knock out mice fibroblast ALKBH4 gene knockout to cause apocyte to produce and the centrosome over-replicate

The fibroblastic propagation of Figure 35 ALKBH4 knock out mice and transfer ability disappearance

Specific embodiment

Embodiment mono-: the experimental technique be applied in the embodiment of the present invention and material

Experiment material:

1, the information of cell line used

2, antibody information used

3, plasmid information used

4, chemical reagent used

Nocodazole(Sigma,M1404),Cytochalasin?D(Sigma,C8273)and?Blebbistatin(Sigma,B0560)，taxol(Invitrogen?P3456)and?phalloidin(Invitrogen?P3457)，Lipofectamine2000(Invitrogen)，Lipofectamine?RNAiMAX(Invitrogen)，PI-stained?kit(Becton-Dickinson)，Annexin?V-FITC&PI?Detection?Kit(Jiamay?Biotech,Beijing,China)，DAPI(Vector?Laboratories,Burlingame,CA)，anti-flag?M2?affinity?gel(sigma,A2220)，anti-HA?affinity?gel(sigma)，GFP-Trap-A?beads(ChromoTek,gta-20)，anti-Rabbit?Ig?IP?Beads(eBioscience)，anti-Mouse?Ig?IP?Beads(eBioscience)，ECL?Western?Blotting?Detection?Kit(GE)

Experimental technique:

1, the structure of expression plasmid

We are with the expression plasmid of conventional sub-clone method construction expression desirable proteins; roughly step is as follows: the method for PCR genes of interest is increased from plasmid or cDNA processing for the design primer; the PCR product is connected with the destination carrier through same enzyme action recycling after specific Cobra venom endonuclease cutting is reclaimed; the connection product proceeds in DH5 α competent cell and increases; the some monoclonals of picking; preliminary enzyme action identifies and connects whether success, through order-checking identify errorless after, amplification for subsequent experimental.Build plasmid primer used in this paper and see experiment material 3.

2, competent cell transforms

1) take out competent cell from-80 ℃ of refrigerators, the bottom of with finger, rubbing gently the EP pipe, melt fast bacterium liquid, and immediately the EP pipe be transferred in ice, standing 10 minutes.

The plasmid that 2) will transform join in 100 μ l competent cells (2-3 μ l plasmid/100 μ l competent cells, the plasmid volume be no more than competent cell 5%), flick EP pipe bottom with finger content mixed; Establish the negative control that only has competent cell and do not add plasmid, ice bath 30 minutes simultaneously.The LB culture fluid can be placed in during this time to 37 ℃ of water-baths and heat, open 42 ℃ of metal baths.

3) the EP pipe is placed in the metal bath of 42 ℃ to standing 90 seconds (not shaking).

4) fast the EP pipe is transferred in ice bath to cooling 2 minutes.

5) every pipe adds 800 μ l LB fluid mediums of 37 ℃ of preheatings, the EP pipe is transferred on the shaking table of 37 ℃ of rotating speed 200rpm to incubation 45 minutes.

6) take out the competent cell that 200 μ l have transformed from the EP pipe, transfer to containing on corresponding antibiotic LB flat board, smoothen with being coated with the bacterium rod.If transforming DNA is to connect product, need centrifugal concentrating cell (centrifugal 10 minutes of room temperature rotating speed 4000rpm), discards 700 μ l supernatant, utilize remaining 200 μ l supernatant re-suspended cells, and transfer evenly is applied to (containing corresponding antibiotic) on LB solid culture flat board.

7) flat board just is being placed in to the incubator of 37 ℃, incubated overnight.

3, protein vivoexpression and purification

The e. coli bl21 that contains expression plasmid (DE3) is seeded in the LB culture medium that contains 100 μ g/ml ampicillin and cultivates in shaking table and increase in 37 degree.When wavelength 600nm light absorption value reaches between 0.5-0.65, add 25 ℃ of inducing culture of final concentration 0.1mM IPTG 4 hours.The centrifugal collection thalline of room temperature rotating speed 4000rpm.Cell suspension is suspended in appropriate lysate, ultrasonication, ultrasound condition is different and different with cracking condition, ultrasonic after, centrifugal 20 minutes of 4 ℃ of rotating speed 16000rpm, supernatant is soluble protein.Before purification, usually to carry out the solubility evaluation.If the destination protein solubility is very low, by changing after condition of culture increases solubility, carry out again purification.

Utilize the protein chromatography system of Bio-Rad to carry out two-step purifying to soluble protein, for the albumen of 6 * His label coupling, the first step, utilize Bio-Scale IMAC cartridge(BIO-RAD 732-4610) carry out affinity purification.Albumen for the coupling of GST label, utilize Bio-Scale Profinity GST cartridge (BIO-RAD, 732-4620) carry out affinity purification, if purity is inadequate, carry out the second step purification: according to the albumen isoelectric point, IP, determine and utilize anion-exchange column (column UNO-Q1, BIO-RAD) or cation exchange column (Column UNO-S1, BIO-RAD) carries out further purification.Before the second step purification and after first step purification, should be dialysed.Usually dialysis twice, each two hours, in the 2L beaker, carry out, how many per sample, select the bag filter (KF134419, Thermo) of different length.The albumen that purification is good finally carries out the SDS-PAGE gel electrophoresis, detects purity and whether meets antibody preparation or other requirement of experiment.

4, SDS-PAGE electrophoresis

SDS-PAGE adopts rectilinear electrophoresis tank device

The preparation of polyacrylamide gel

1), the preparation of gel slab

A. wash plate:

Clean two glass plates with liquid detergent, then clean with tap water, then with 75% ethanol, that blackboard eraser is dry.

B. matching board:

Two glass plates are stacked neatly, clip with the clip both sides, these two glass plates are fixed on base.Insert supporting comb, in the line of comb lower edge, with waterproof marking pen labelling encapsulating position.Take out comb.

2), encapsulating:

B. encapsulating: utilize pipet that separation gel (avoiding Bubble formation) is splashed between two glass plates after above-mentioned solution is mixed, reach comb lower edge 1cm place to liquid level.Slowly add ddH with pipet ₂the O sealing, note the not random glue face of communications centre.Standing, after glue polymerization to be separated, go or suck water layer with filter paper.It can be wrapped to preservative film and put into 37 ° of constant incubator 30min.

C. join concentrated glue (5%) solution 6ml:

At once add concentrated glue (avoiding Bubble formation) with pipet after above-mentioned solution is mixed and be overlying on two separation gels between glass plate to full, insert gently comb.It is wrapped to preservative film and put into 37 ° of constant incubator 30min.After it condenses, make gel slab.Perform mark with pen at the intersection of separation gel and concentrated glue, later encapsulatings according to this all.Make 2 clotting offset plates.

3), sample treatment:

A. by-20 ℃ of frozen cell pellet, every pipe equal-volume adds 50 μ l 1 * SDS loading buffer, is placed on incubation 5min in 37 ℃ of water-baths.

B.95 ℃ constant-temperature metal bath heats 15min.

C. with 13,300rpm high speed centrifugation 5min.Shift supernatant to new EP pipe, carry out labelling.

D. get three EP pipes, wherein a pipe adds 10 μ l marker and 10 μ l 1 * SDS loading buffer, mixes standby; One pipe adds 2 μ l marker and 8 μ l 1 * SDS loading buffer, mixes; Another pipe adds 10 μ l 1 * SDS loading buffer.

4), loading;

A. the clip on the gel slab of two glass plates being made sheds.Take out lightly the comb in gel slab.

B. gel slab is vertically leaned against on the power bay in electrophoresis tank, make the recessed of gel slab rely on power bay along face.Common two clotting offset plates share a power bay.

C. gel slab and power bay are fixed in power slot on request.On request respectively toward in two clotting offset plate intermediate power supplies framves and add 1 * Tris-Glycine electrophoretic buffer of proper volume in electrophoresis tank, do not have gel to liquid level.

D. get the sample liquid after processing, with liquid-transfering gun, draw 10 μ l sample liquid, by sample liquid slowly add in gel slab recess position (sample is clicked and entered place).Note not breaking up sample.

5), electrophoresis:

Connect electrophoresis tank and electrophresis apparatus with two wires, notice that red plug and socket with black electrodes matches.Switch on power.Control voltage is 70V in concentrated glue, about 30min, and after the concentrated glue of passing by, voltage is adjusted to 120V, approximately 1h.When the band of sample bromophenol blue during electrophoresis on the observation gel slab is walked out separation gel, stop electrophoresis.Powered-down.Then carefully take out gel slab in electrophoresis tank.

6), dyeing

A. with blade or thin plate by gel slab two outer glass plates pry open gently, gel is pitched therein on a glass plate.

B. use blade junction along separation gel and concentrated glue on gel, first will concentrate glue and cut and abandon, then separation gel is cut, and cut away Yi little Jiao in the upper left corner of separation gel, with labelling sample order.

C. then separation gel is carefully moved in staining utensil.

D. add 50ml dyeing liquor (about 5 times of volumes) in staining utensil, preservative film sealing, shaking table dyeing 3h.

7), decolouring

Dyeing liquor is gone.The water rinse number time for gel of dyeing, drain the water and be placed in staining dish, then add the 100ml destaining solution, adds a cover, till the shaking table decolouring can clearly demonstrate protein band to the gel dyeed after decolouring.

5, Western hybridization technique

Usually we the sample degeneration is good after on 10%SDS-PAGE glue electrophoresis, albumen is transferred on pvdf membrane by half-dried transferring film instrument.Through primary antibodie and two anti-labellings, utilize the colour developing of the ECL of GE company chemical luminous substrate, finally with the colour developing of X-ray egative film.

1) carry out electrophoresis according to the method for above-mentioned SDS-PAGE, but just with a clotting glue.

2) transferring film

A. cut one and PAGE glue sizable Immobilon-P Transfer Membrane and 4 3mm Whatman Chromatography paper.First Immobilon-P Transfer Membrane is dipped in to about 30min in methanol, then soaks in Transfer buffer.Sponge and Whatman Chromatography paper also can be dipped in to Transfer buffer during this period.

B. make electricity and turn sandwich, order is as follows:

Positive level (white or other light) sponge

2 Whatman Chromatography paper

Immobilon-P?Transfer?Membrane

PAGE glue

2 Whatman Chromatography paper

Negative level (black) sponge

After being overlying on glue, with scoop, drive bubble away to film.

3) the sandwich clip is put into to the electrophoresis tank of the Transfer Buffer that is preinstalled with pre-cooling on ice, be sidelong into ice chest at negative pole one, add a cover, switch on power.Constant voltage 100V, 1h.

4), after transferring film completes, first Immobilon-P Transfer Membrane shaking table in 1 * TBST wash buffer is soaked to 5min.

5) Immobilon-P Transfer Membrane is immersed in containing in 1 * TBSTwash buffer of 5% skim milk, deposits in 4 ℃ of refrigerator overnight.

6) Immobilon-P Transfer Membrane is placed in the plastic bag of three side sealing mouth, add 5ml to contain 1 * TBST wash buffer of 5% skim milk and the mixed liquor of 2.5 μ l 6 * His tag antibody, with pipet, rotate gently and drive bubble away.Be placed on shaking table and hatch 1h.

7) cut off plastic bag Immobilon-P Transfer Membrane is taken out, with 1 * TBSTwash buffer, clean 3 * 10min.

8) preservative film is flattened and is laid on laboratory table, with tweezers, the edge of film is contacted to a handkerchief paper gently water is drained, be laid on preservative film.

9) get a 15ml centrifuge tube, respectively get 1ml Detection reagent1 and Detection reagent2, mix.

10) use the above-mentioned mixing drop of pipette, extract on Immobilon-P Transfer Membrane, timing 1 ~ 5min.

11) with preservative film, Immobilon-P Transfer Membrane is wrapped up, be placed in a film cassette.

12) (lamp is turned off) in darkroom, taken out a slice film, cut one jiao on upper left side, be placed on the glue in film cassette, attention will make film only depend on the edge of film cassette, builds lid, exposure 1min.

13) take out film, be put on developing machine and develop photographic film.

14) take out again second new unexposed film, estimate the time of current exposure according to the effect of first film rinsing, expose for the second time.

15) make a record on film, with the marker image of standard, do contrast, estimate the molecular size range of the albumen of expression.

6, prepared by the protein sample for mass spectral analysis

About 5 * 10 ⁷the about 10mg total protein of the 293T cell lysate of stably express Flag-GFP and Flag-GFP-ALKBH4 is for the preparation of protein sample.The method of application immunoprecipitation, pearl (the anti-FLAG M2 agarose of cell pyrolysis liquid and 100ul coupling FLAG antibody, sigma) hatch 5 hours for 4 ℃, again by the TBS buffer solution for cleaning that contains 250mMNaCl three times, glycine (glycine) eluting that to be combined on pearl with the albumen of FLAG label be 100mM with the final concentration of pH 3.5.The albumen eluted separates through SDS-PAGE, and silver dyes.Cut specific band albumen, send company to do the LC-MS/MS mass spectral analysis.

7, cell culture

Freeze-stored cell recovery: in advance by 37 ℃ of water-bath modulation, take out frozen cell and put into rapidly 37 ℃ of water-baths from liquid nitrogen container, then in water-bath, constantly shake cryopreservation tube, until cells frozen storing liquid just melts.By cell sucking-off from cryopreservation tube, put into the centrifuge tube that adds in advance culture medium with aseptic straw, room temperature rotating speed 1000rpm removes supernatant in centrifugal 3 minutes, finally uses complete medium resuspended cell, puts into 37 ℃ of incubators and cultivates.

Cell culture: the culture medium of MRC5 cell, 293T cell, OSC-20 cell and TOC2 cell is for containing 10% calf serum, 100U/ml two anti-(mycillins), nonessential aminoacid, the RPMI1640 culture medium of 1mM Sodium Pyruvate (Invitrogen, 31800022).The U2OS cell is used the DMEM high glucose medium to cultivate, and other compositions are identical.All cells, all at 37 ℃, is cultivated containing in the 5%CO2 incubator.

Passage: first suck culture fluid in culture dish, PBS washes once, add pancreatin at 37 ℃ of peptic cells, after seeing cell rounding or cell detachment under microscope, with complete medium, stop digestion, the about 3-10 minute of digestion process, the concrete time determine according to cell type, blows and beats and several times cell is dispelled gently, take out a certain amount of cell in new culture dish for other experiment, unnecessary cell sucking-off is abandoned in the waste liquid cylinder, in culture dish, add fresh culture medium to continue to cultivate.

Cell cryopreservation: RPMI1640 or DMEM cryopreserving liquid that preparation contains 10%DMSO and 20% calf serum, put into 4 ℃ of refrigerator pre-coolings, after attached cell is digested to centrifugal collection, cryopreserving liquid re-suspended cell with prior 4 ℃ of pre-coolings, by cryopreservation tube of each diameter 10cm culture dish cell packing, cryopreservation tube is put in the program freezing storing box that isopropyl alcohol is housed, be placed in-80 ℃ of refrigerator overnight, be saved in liquid nitrogen container next day.

8, the foundation of stable cell lines

In the present invention, respectively with 293T, the MRC5 cell is recipient cell, has built respectively the stable expression cell line of stably express GFP albumen, GFP-ALKBH4 albumen, 3 * Flag albumen, 3 * Flag-ALKBH4 albumen, HA albumen, HA-β-actin albumen.Process of establishing is as follows: first recombiant plasmid is used to the restricted enzyme linearisation, the selected restriction enzyme site principle of linearisation: do not affect the expression of its destination protein and resistant gene in eukaryotic cell after plasmid linearization, and the linearization plasmid purification is quantitative.Reach 60%-70% at first 24 hours of transfection inoculation right quantity cell when making it in transfection in six orifice plates, and with not containing antibiotic culture medium culturing.By 4 μ l Lipofectamine 2000 (Invitrogen, 11668-027) He 2 μ g purpose plasmids are respectively at culture medium OPTI-MEM(31985, the Gibco of 250 μ l serum-frees) mix, after static 5 minutes, both mixing are shaken up, and static 15 minutes.Suck during this time former culture medium, wash and once add again the antibiotic OPTI-MEM culture medium of 500 μ l serum-free in six orifice plates with PBS, quiescent time to after plasmid and Lipofectamine 2000 mixture are added in six orifice plates, do not change into after 4 hours containing antibiotic culture medium and continue to cultivate.Approximately after 24 hours, add neomycin G418 screening positive cell, approximately about ten days, the stable cell lines containing the GFP fluorescin screens positive cell with flow cytometer, the positive cell of some is continued to cultivate, after immunoblot experiment checking protein expression, a part is frozen, and a part is for subsequent experimental.Till the stable cell lines of other label protein screens a large amount of amplifications with G418, next step is frozen or for follow-up test.

9, immunofluorescence technique

1) cell of the about 30-40% of inoculation in six orifice plates, add medicine or transfection DNA, RNA next day, after 48 hours, receives cell.

2) with PBS, wash twice.

3) fixing: each hole adds the methanol of 1ml-30 ℃ of refrigerator pre-coolings, fixes approximately 10 minutes on ice.

4) infiltration punching: PBS washes three times, adds 1ml 0.1%TritonX-100 and 0.5%NP-40 to permeate processing to cell, hatches on ice 10 minutes.

5) sealing: after PBS washes three times, with the sealing of the TBST solution containing 5% skim milk 30 minutes.

6) coverslip is taken out and is placed in six orifice plates that are covered with sealed membrane, add the primary antibodie containing 5% defatted milk powder TBST dilution for 100 μ l, hatch one hour for 37 ℃.

7) coverslip is taken out and is placed in six orifice plates that there is no sealed membrane, clean three times each 5 minutes with TBST.

8) coverslip is taken out and to be placed in six orifice plates that are covered with sealed membrane, add 100 μ l anti-with the fluorescence two containing 5% defatted milk powder TBST dilution, place it in magazine 37 ℃ and hatch half an hour.

9) coverslip is taken out and is placed in six orifice plates that there is no sealed membrane, clean three times with TBST, each 5 minutes, then wash once with PBS.

10) finally with the mountant transfect cell core that contains DAPI (Vetor H-1200), with transparent nail polish mounting.With Leica TCSSP5 confocal fluorescent microscope, celluar localization is observed.

10, Immunoprecipitation

1) cell of the about 30-40% of inoculation in the 10cm culture dish, transfection next day plasmid, 48 as a child after collecting cell, centrifugal, PBS cleans twice.

2) add the long-pending 0.1%NET cell pyrolysis liquid of cell precipitation pentaploid, place on ice 30 minutes.

3) ultrasonic degradation cell, be placed on the EP pipe on ice and, with cursory fixing, ultrasound condition: 10% output, ultrasonic time 1 minute, each ultrasonic 10 seconds, stop 20 seconds.

4) centrifugal in 4 ℃ of centrifuges after ultrasonic, centrifugal 10 minutes of rotating speed 14800rpm.Take out supernatant solution, with the quantitative albumen of Bradford, take out 200 μ g as total protein, add 5 * the albumen sample-loading buffer, 95 ℃ of degeneration 10 minutes.

5) get the anti-Flag of 35 μ l, HA or GFP pearl and be added in 1.5ml EP pipe, with containing the TBS buffer solution for cleaning of 150mM NaCl three times.

6) get the 1.5mg total protein and be added in the pearl cleaned, then add 0.1%NET lysis buffer polishing to 1ml, the EP pipe is put on rotary incubator to 4 ℃ and hatches 5 hours.

7) with centrifugal 1 minute of 4 ℃ of centrifuge speed 5000g, supernatant discarded, the TBS buffer that adds 1ml to contain 150mM NaCl is put into rotary incubator and cleans 5 minutes, centrifugal 1 minute of 4 ℃ of rotating speed 5000g, supernatant discarded, repeat to wash three times.

8) after centrifugal supernatant discarded, more once centrifugal, with syringe, liquid is blotted only, add 30 μ l 2 * albumen sample-loading buffers and 70 μ l1 * albumen sample-loading buffer, mix, 95 ℃ of degeneration 10 minutes.For SDS-PAGE, follow-up silver dye, the Western experiment.

11, plasmid transfection

1) the previous day is inoculated 30% cell in 10ml antibiotic-free culture medium in transfection in the 10cm culture dish.

2) for each transfection hole, dilute 5 μ g plasmids in 500 μ l serum-frees and antibiotic OPTI-MEM culture medium, mix, the PEI that gets 30 μ l1mg/ml is added to the inside, mixes static 15 minutes of room temperature.

3) culture medium in culture dish is absorbed; with PBS, clean once; add again 4.5ml serum-free and antibiotic OPTI-MEM culture medium; time to after the plasmid mixture of front is added in cell; being put into 37 ℃ of incubators continues to cultivate; 4-6 as a child absorbed containing the transfection reagent culture medium, changed fresh complete medium and continued to cultivate, and after 48 hours, detected.

12, siRNA transfection

1) for the mRNA sequence of people ALKBH4, we entrust Shanghai Ji Ma company to design and synthesize the siRNA of three rules for different target spots.

2) the previous day is inoculated 30% cell in 2ml antibiotic-free culture medium in transfection in six orifice plates.

3), for each transfection hole, dilution 60pmol siRNA, in 250 μ l serum-frees and antibiotic OPTI-MET culture medium, mixes.Add again 6 μ l Lipofectamine RNAiMAX (Invitrogen, 13778150), mix, static 15 minutes of room temperature.

4) in the quiescent period, culture medium in six orifice plates is absorbed, with PBS, clean once, add 750 μ l serum-frees and antibiotic OPTI-MEM culture medium in the transfection hole.

5) after arriving quiescent time, mixture is added in the transfection hole.Cultivate in 37 ℃ of incubators and within 4-6 hour, change into not containing antibiotic complete medium.After 48 hours, experiment detects.

13, Flow Cytometry detects cell cycle

1) the previous day is inoculated 30% cell in 10ml antibiotic-free culture medium in transfection in the 10cm culture dish.After 24 hours, transfection siRNA, change liquid in 4-6 hour, puts 37 ℃ of incubators and cultivate.

2) digest collecting cell after 48 hours, centrifugal 5 minutes of room temperature 1000rpm, wash once with PBS, use again buffer solution in cell cycle test kit (CycleTest Plus DNA regentkit, BD, Cat:340242) to clean once, centrifugal, abandon supernatant.

3) each sample adds 100 μ l buffer A, hatches 10 minutes for 37 ℃.

4) add 100 μ l buffer B, hatch 10 minutes for 37 ℃.

5) add 100 μ l buffer C, lucifuge incubated at room 10 minutes.

6) flow cytometer detects.

14, Flow Cytometry detects M phase cell

1) the previous day is inoculated 30% cell in 10ml antibiotic-free culture medium in transfection in the 10cm culture dish.After 24 hours, transfection siRNA, change liquid in 4-6 hour, puts 37 ℃ of incubators and cultivate.

2) digest collecting cell after 48 hours, centrifugal 5 minutes of room temperature rotating speed 1000rpm, wash once with PBS, then, with adding 1ml4% paraformaldehyde fixed cell, fix 10 minutes on ice.

3) add 10ml and wash once containing the PBS of 1%FBS, centrifugal 5 minutes of room temperature rotating speed 1000rpm, abandon supernatant.

4) slowly add 90% methanol of 1ml-30 ℃ of pre-cooling, make the Premeabilisation of cells perforation, be placed on static 30 minutes on ice.

5) add 10ml and wash once containing the PBS of 1%FBS, centrifugal 5 minutes of room temperature rotating speed 1000rpm, abandon supernatant.

6) add 100 μ l 1%BSA sealings, 37 ℃ are sealed 30 minutes.

7) room temperature room temperature 1000rpm is centrifugal 5 minutes, abandons supernatant, adds the primary antibodie of the anti-HistoneH3-S10P that 100 μ l have diluted with 1%BSA, at 37 ℃, hatches 1 hour.

8) room temperature 1000rpm is centrifugal 5 minutes, abandons supernatant, adds 10ml PBST and cleans, the centrifugal supernatant of abandoning.

What 9) add that 100 μ l have diluted with 1%BSA is two anti-, at 37 ℃, hatches 30 minutes.

10) room temperature 1000rpm is centrifugal 5 minutes, abandons supernatant, adds 10ml PBST and cleans, the centrifugal supernatant of abandoning.

11) with 200 μ l PBS re-suspended cells, then add the RNase A that 10 μ l concentration are 10mg/ml, mix, hatch 30 minutes at 37 ℃.

12) add propidium iodide (PI) solution that 20 μ l concentration are 1mg/ml, lucifuge is hatched 10 minutes.

13) flow cytometer detects.

15, Flow Cytometry detects apoptosis

1) the previous day is inoculated 30% cell in 10ml antibiotic-free culture medium in transfection in the 10cm culture dish.After 24 hours, transfection siRNA, change liquid in 4-6 hour, puts 37 ℃ of incubators and cultivate.

2) digest collecting cell after 48 hours, centrifugal 5 minutes of room temperature rotating speed 1000rpm, wash once with PBS,

3) next use the apoptosis test kit Annexin V-FITC&amp of Beijing Jiamei company; PI Detection Kit labelling apoptotic cell, the kit method operation is pressed in back.

4) flow cytometer detects.

16, separation of shrink ring and middle body protein.

1) at the MRC5 Growth of Cells during to 50% left and right, add the thymidine (thymidine) that final concentration is 2mM to cultivate 16 hours, wash once with PBS again, add fresh culture continue to cultivate 4-5 hour, then add the nocodazole that final concentration is 0.1 μ g/ml and continue to cultivate 5 hours.

2) the drug treating time starts to collect division cells after reaching, cell in division stage can shrink and become round adherent insecure, so our employing is rocked culture bottle the cell of division stage is rocked, there are 37 ℃ of fresh culture medium to wash twice the division cells of collecting, add 37 ℃ of fresh culture medium to continue to cultivate 0.5-1 hour, purpose is in order to make cell in the cytokinesis phase again.

3) cell obtained is divided into to two parts, a part is extracted total protein with the cracking of protein cleavage buffer, another part adds taxol and the phalloidin that final concentration is 5 μ g/ml to stablize spindle microfilament micro-tubular structure, hatch 0.5-1 minute, centrifugal 1 minute of room temperature rotating speed 1000rpm, supernatant discarded, slowly add aquesterilisa to clean cell along tube wall, sucks moisture content.

4) add the hypotonic lysis buffer (2mM Pipes at pH6.9,0.25%Triton X-100,20ug/ml taxol) of ten times of cell precipitation volumes, re-suspended cell, incubated at room 20 minutes,

5) room temperature rotating speed 2000rpm is centrifugal 20 minutes, and supernatant discarded, be placed on cooled on ice.

6) add 1ml 50mM pH6.3MES lysis buffer is cleaned up, then use the resuspended precipitation of 100 μ l 50mM pH6.3MES, resuspended precipitation is layered on 40% glycerol, centrifugal 20 minutes of room temperature rotating speed 2000g, be precipitated albumen.Add 50 μ l albumen sample-loading buffers, 95 ℃ of Denatured proteins.

Embodiment bis-: purification ALKBH4 fusion rotein prepares anti-ALKBH4 albumen rabbit source antibody

1, ALKBH4 protein structure domain and possible active mechanism

Escherichia coli AlkB albumen is monokaryon non-heme Fe ²⁺dioxygenase superfamily member with the α-ketoglutaric acid dependence, it be take metal ion and take oxygen molecule as cosubstrate as prothetic group, activate the contacted Me of nitrogen-atoms on dioxygen Journal of Molecular Catalysis oxidation and stable heterocycle, make it conformation and change, thereby remove Me by the form of release formaldehyde.ALKBH albumen is human dioxygenase AlkB protein family a member, and the general character of this family protein is to have escherichia coli AlkB protein function domain: the ferrous ion binding structural domain is HxDx _nthe H domain, α-ketoglutaric acid and Binding Capacity domain are the RxxxxxR domain.And ALKBH4 albumen also has such domain: iron ion binding structural domain H169-D171-H254, α-ketoglutaric acid and Binding Capacity domain R265-R271, as shown in Figure 1.Histone demethylase JHDM family protein also belongs to the dioxygenase albumen of iron ion and α-ketoglutaric acid dependence, they are specific protein demethylases, and dioxygenase AlkB family protein has identical demethylation mechanism with the JHDM family protein, as shown in Figure 2, the effect substrate of this explanation AlkB family protein may be also protein, and ALKBH4 albumen is potential protein demethylase.

2, purification 6 * His-ALKBH4 wild type and mutant fusion protein.

According to the domain of ALKBH4 and the demethylation mechanism similar to histone demethylase, we predict that ALKBH4 albumen has the activity of protein demethylation, therefore to study the activity of ALKBH4, at first to analyze ALKBH4 albumen biochemical activity in vitro, we have built the recombiant plasmid pDEST17-ALKBH4 of escherichia coli expression, he can express 6 * His-ALKBH4 fusion rotein, the people ALKBH4cDNA sequence that expression obtains is shown in sequence table SEQ ID NO.1, and the people ALKBH4 protein sequence that translation obtains is shown in sequence table SEQ ID NO.6.We use the rite-directed mutagenesis test kit of Stratagene company to build pDEST17-ALKBH4 H169A-D171A-H254A mutant plasmid, point mutation primer used has: H169A-D171A mutant primer SEQ ID NO.18 and two point mutation primers of H254A mutant primer SEQ ID NO.19, the people ALKBH4 HDH mutants cDNA sequence that expression obtains is shown in sequence table SEQ ID NO.2, and the people ALKBH4HDH mutant protein sequence that translation obtains is shown in sequence table SEQ ID NO.7.

Purification 6 * His-ALKBH4 wild-type protein and HDH mutant fusion protein albumen: wild type and mutant recombinant plasmid transformed are in protease-deficient BL21 competent cell, be coated with dull and stereotyped, the picking monoclonal, be seeded in the LB culture medium that contains 100 μ g/ml ampicillin, cultivate amplification in 37 degree shaking tables.When wavelength 600nm place light absorption value reaches between 0.5-0.6, add 25 ℃ of inducing culture of final concentration 0.1mM IPTG 4 hours.At 4 ℃ of centrifugal collection thalline of rotating speed 4000rpm, bacterial precipitation is resuspended in the long-pending lysate of decaploid, ultrasonication, ultrasound condition is different and different with cracking condition, ultrasonic after, centrifugal 20 minutes of 4 ℃ of rotating speed 16000rpm, supernatant was soluble protein.

Utilize the quick protein purification system of Bio-Rad to carry out purification to soluble protein, albumen for 6 * His label, the first step, utilize Bio-Scale IMAC cartridge(BIO-RAD 732-4610) carry out affinity purification, dye to identify again the purity of gained albumen with SDS-PAGE electrophoresis and Coomassie brilliant blue, purification result as shown in Figure 3.In figure, M represents the molecular weight of albumen standard substance, and S represents soluble protein, and F represents the composition that on soluble protein, complete pillar flows out, digitized representation collecting pipe numbering.

From result, can find out, the collecting pipe purity of front is not high, and the purity of back is high, so we are collected together the 12-20 pipe of wild-type protein, and the 8-12 pipe of mutant is collected together, and with Bradford reagent quantitative albumen, finds that concentration is lower.Next step uses albumen concentration tube protein concentrate, and the albumen concentration tube that we use is VIVASPIN 20 concentration tubes of Sai Duolisi company, can hold back the above protein molecular of 10KD, so not only can protein concentrate but also can remove small molecular weight impurity.Protein quantification after concentrated also detects purity with SDS-PAGE electrophoresis, coomassie brilliant blue staining, immunoblot experiment, and as shown in Figure 4, Fig. 4 a is the coomassie brilliant blue staining result to result, Fig. 4 b, and c is the immunoblot experiment result.Result shows that we surpass 95% by resulting purity of protein, have reached the purity of biochemical analysis.

3, purification GST-ALKBH4 fusion rotein reaches the antibody for the preparation of immunofluorescence experiment

The function antibody of an albumen of research is very crucial experiment material, owing to there is no business-like ALKBH4 antibody, so we want own Dispersal risk, at first we will study ALKBH4 at thin inner cellular localization and need look for ALKBH4 effect substrate and interaction factor thereof, so just need one of preparation can identify the antibody of the three-dimensional epi-position of ALKBH4 molecule, we just need one of purification to have the ALKBH4 molecule of nature conformation as immunogen, because the purity of 6 * His-ALKBH4 fusion rotein of purification is before done the standard of antibody not, therefore we select GST label plasmid as expression system.At first we build the pGEX-5X-2-ALKBH4 recombiant plasmid, select restriction enzyme site, the design primer, forward primer is SEQ ID NO.11, reverse primer is SEQ ID NO.12, extract mRNA from the 293T cell, reverse transcription PCR amplification ALKBH4 gene, the ALKBH4 cDNA sequence increased is SEQ ID NO.1, enzyme action connects, choose monoclonal upgrading grain order-checking, verify errorless after, the amplification plasmid.The ALKBH4 protein sequence that plasmid expression obtains is SEQ ID NO.6.

Purification GST-ALKBH4 wild type fusion rotein: at first will carry out the solubility evaluation, recombinant plasmid transformed is in the BL21 competence antibacterial of protease-deficient, be coated with dull and stereotypedly, the picking monoclonal, be seeded in the LB culture medium that contains 100 μ g/ml ampicillin amplification cultivation in 37 ℃ of shaking tables.When wavelength 600nm light absorption value reaches between 0.5-0.6, add 25 ℃ of inducing culture of 0.1mM IPTG 4 hours.The centrifugal collection thalline of room temperature rotating speed 4000rpm.Bacterial precipitation is resuspended in the long-pending lysate of decaploid, ultrasonication, ultrasonic rear taking-up 1/5th is as total protein, centrifugal 20 minutes of 4 ℃ of rotating speed 16000rpm, supernatant is soluble protein, again total protein (T), supernatant (S) and precipitation (P) are added to the albumen sample-loading buffer, 95 ℃ of degeneration 10 minutes.With the BL21 of Pignus pignoris grain not in contrast, processing method as above.The sample processed, in two groups of samples on identical ratio, is done to SDS-PAGE electrophoresis coomassie brilliant blue staining for one group, and another group is done immunoblot experiment checking solubility and expression.As shown in Figure 5 a, the GST-ALKBH4 fusion protein expression is high and solubility is fine for result.

Next step utilizes the quick protein purification system of Bio-Rad to carry out purification to soluble protein, albumen for the GST label, the first step, utilize Bio-Scale Profinity GST cartridge (BIO-RAD, 732-4620) carry out affinity purification, the purifying protein amount is very large but purity is not high as a result, as shown in Figure 5 b.Now purity of protein is fewer than Dispersal risk, therefore lower step will take ion-exchange chromatography to be further purified, the isoionic point pI value that software analysis obtains the GST-ALKBH4 fusion rotein is 6.86, approaching neutral ion exchange effect is difficult to determine, we carry out further purification by choice for use cation exchange column (ColumnUNO-S1, BIO-RAD).Purification effect is fine as a result, and the purity of protein of all collecting pipes has all reached the standard of making antibody, and purity reaches more than 98%, and purification result as shown in Figure 5 c.In figure, M represents protein standard substance, and S represents soluble protein, and F represents the composition that on soluble protein, complete pillar flows out, digitized representation collecting pipe numbering.Concentrate with VIVASPIN 20 concentration tubes of Sai Duolisi company after gained albumen is collected to mixing, deliver to the system in Beijing Jing sky and become Bioisystech Co., Ltd's Dispersal risk.Antibody purification: the rabbit that company completes immunity kills blood-letting, centrifuging and taking serum, the ProteinA Sepharose affinitive layer purification rabbit source antibody of use sigma company.We add the Hydrazoic acid,sodium salt of 1.5M to make final concentration reach 15mM the ALKBH4 antibody obtained, and adding the Hydrazoic acid,sodium salt purpose is in order to prevent that antibody is by bacterial decomposition.

Antibody checking: use the reticent ALKBH4 gene of siRNA in the MRC5 cell, concrete RNA silencing methods is shown in embodiment mono-, ALKBH4siRNA sequence used is shown in sequence table SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, we reduce the ALKBH4 protein expression by these three siRNA sequence mixing transfectional cells.Adopt the specificity of immunofluorescence method detection ALKBH4 antibody again, find the middle body structure and the centrosome that are positioned at cytokinesis of this antibody specificity, as shown in Figure 7, follow-up also have immunofluorescence experiment checking ALKBH4 in intracellular location.

4, for the preparation of the rabbit source ALKBH4 antibody of immunoblot experiment

Prepare the antibody that can be used in immunoblot experiment, in order to improve the specificity of antibody, polypeptide is optimum selection as immunogenic antibody.We cooperate with Kang Wei ShiJi Co., Ltd, the upper much higher peptide 101-QSGRKKQDYGPK-112 of immunogenicity of the synthetic ALKBH4 of company, as shown in Figure 8 a.Three immune rabbits, concrete experiment process and use reagent company maintain secrecy.The good rabbit that kills of immunity is got the centrifugal acquisition serum of blood, with the Protein A antibody purification of Sigma company.The good antibody by purification, with the specificity of immunoblot experiment checking ALKBH4 antibody.The GST-ALKBH4 albumen of front purification and GST albuminous degeneration are processed, upper two groups of sample SDS-PAGE electrophoresis, transferring film, use respectively ALKBH4 antibody and GST antibody hybridization checking, found that ALKBH4 antibody can identify GST-ALKBH4 albumen, although below several assorted bands are arranged, find it is the albumen of degraded after the hybridization of GST antibody, so this antibody can be for immunoblot experiment, as shown in Figure 8 b.We also detect ALKBH4 antibody specificity and protein expression level in eukaryotic cell, use ALKBH4 siRNA reticent ALKBH4 gene in the MRC5 cell, ALKBH4 siRNA sequence used is shown in sequence table SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, cell lysis extracts albumen and does the immunoblot experiment detection, found that in the cell of transfection siALKBH4, the ALKBH4 expression is starkly lower than matched group, the ALKBH4 albumen of the identification eukaryotic cell expression that as shown in Figure 8 c, this explanation ALKBH4 can be specially.

Sum up, our purification 6 * His-ALKBH4 wild type and the HDH mutant fusion protein for biochemical activity, analyzed, for next step, to analyze the ALKBH4 protein active ready.Also prepared can be for two kinds of antibody of immunofluorescence experiment and immunoblot experiment simultaneously, and by the gene silencing experimental verification specificity of these two antibody, for the functional study of back lays the first stone.

Embodiment tri-: checking ALKBH4 gene is in intracellular location and expression

1, the ALKBH4 protein localization is on centrosome (centrosome).

This experiment verifies that by immunofluorescence technique ALKBH4 is at thin inner cellular localization, the MRC5 cell is inoculated in six orifice plates that are covered with coverslip, inoculum density 30-40%, add evening next day the Nocodazole of final concentration 0.04g/ml to process 16 hours, adding the Nocodazole purpose is to make cell block in mitotic phase.Wash once with PBS after 16 hours, change fresh complete medium into and continue to cultivate 1 hour, PBS washes twice, fixing infiltration punching, and concrete grammar is with embodiment mono-.Primary antibodie is used centrosome label γ-tubulin and spindle label α-tubulin and ALKBH4 antibody jointly to hybridize, and the anti-Mus two of two anti-use FITC couplings resists the anti-hybridization of anti-rabbit two with the Cy3 coupling.Use Laser Scanning Confocal Microscope, take the cell cycle ALKBH4 celluar localization in each period, from the interphase cell to the mitosis telophase, see in result that ALKBH4 locates altogether with centrosome label γ-tubulin each period, as shown in Fig. 9 a, this explanation ALKBH4 protein localization is at centrosome.Simultaneously we also find the anaphase of cell division and latter stage, ALKBH4 albumen is at retraction ring (contractile ring) and middle body (midbody) is upper locates, and as shown in Fig. 9 b, below will describe in detail.But do not find that ALKBH4 and spindle label α-tubulin locate altogether, just locate altogether on middle body in mitosis telophase, illustrate that the ALKBH4 delocalization is on spindle, as shown in Fig. 9 b.

In order further to verify ALKBH4 albumen, on centrosome, locate, we build MRC5/3 * Flag and MRC5/3 * Flag-ALKBH4 stable cell lines, purpose identifies that the ALKBH4 of heterogenous expression is in intracellular location, at first build the ALKBH4 plasmid of 3 * Flag label, the primer is shown in sequence table forward primer SEQ ID NO.14 and reverse primer SEQ ID NO.15, amplification obtains people ALKBH4 cDNA and sees sequence table SEQ ID NO.1, expresses people ALKBH4 wild-type protein and sees sequence table SEQ ID NO.6.Also build the ALKBH4H169A-D171A-H254A mutant plasmid of 3 * Flag label simultaneously, point mutation primer used has: H169A-D171A mutant primer SEQ ID NO.18 and two point mutation primers of H254A mutant primer SEQ ID NO.19, and express people ALKBH4 HDH mutant protein and see sequence table SEQ ID NO.7.Again by the plasmid transfection MRC5 cell construction stable cell lines built.We hybridize jointly with Flag and γ-tubulin antibody, and with the anti-hybridization of corresponding fluorescence two.The ALKBH4 albumen that found that external source is also located altogether with γ-tubulin, and the cell of expressing 3 * Flag empty carrier location altogether not, as Figure 10 a just further proof ALKBH4 be positioned on centrosome.We have also adopted the method separation center body of sucrose concentration gradient centrifugation, and detect the expression of ALKBH4 in centrosome by the immunoblot experiment method, the intrinsic albumen γ-tubulin of the centrosome of usining and CEN1 are as positive control, find that ALKBH4 expression trend and CEN1 are same as γ-tubulin identical, as shown in Figure 10 a, this as a result let us be sure of that the ALKBH4 protein localization points out and be the proper constituent of centrosome at center.

2, ALKBH4 is positioned on retraction ring (contractile ring) and middle body (midbody).

The immunofluorescence experiment of front is tentatively found ALKBH4 when mitosis anaphase and cytokinesis and α-tubulin locates altogether, and as shown in Fig. 9 b, we guess that ALKBH4 is positioned on middle body.In order to verify conjecture, use chemical markers phallotoxins-Texas Red and the ALKBH4 antibody cohybridization cell of F-actin, F-actin can the visible marking go out retraction ring and middle body structure, found that ALKBH4 albumen and F-actin have significantly location altogether on retraction ring and middle body in the MRC5 cell, as shown in Figure 11 a, external source 3 * flag-ALKBH4 also has significantly location altogether on retraction ring and middle body in F-actin simultaneously, as shown in Figure 11 b.Again another molecular marked compound anillin of ALKBH4 antibody and retraction ring and middle body is done to immunofluorescence dyeing jointly, result shows that ALKBH4 and anillin locate fully altogether on retraction ring and middle body, as shown in Figure 11 c.These result proofs ALKBH4 is positioned on retraction ring and middle body.

For checking ALKBH4 molecule is retraction ring and middle body component, we use chemical method separation of shrink ring and middle body protein from the MRC5 cell, stablize spindle microfilament microtubule with taxol and phalloidin, hypotonic processing cell lysis, obtain retraction ring and middle body protein, whether western blotting method checking ALKBH4 expresses in the above, we use retraction ring and middle body tag albumen NM II, Plk1, actin and α-tubulin are as positive control albumen, p53 is as negative control albumen, result is as shown in Figure 11 d, in the retraction ring that ALKBH4 and positive control molecule all are separated at us, in the body component, express, and negative control molecule p53 does not express in the above, this illustrates our separation method success, and proof ALKBH4 is retraction ring and middle body protein.

Sum up, immunofluorescence experiment proof ALKBH4 is positioned on centrosome, retraction ring and middle body, and centrosome and retraction ring and middle body separating experiment proof ALKBH4 are the components of these organelles.

Embodiment tetra-: checking ALKBH4 interaction protein Pseudobulbus Bletillae (Rhizoma Bletillae) effect substrate

1, research ALKBH4 latent effect substrate or interaction factor

Learn that in the face of result ALKBH4 is positioned on centrosome, retraction ring and middle body in the past, so we predict that ALKBH4 is relevant to cell division, may participate in the demethylation of some albumen in fission process.Although existing article shows that ALKBH4 albumen has biochemical activity in testing in vitro, its concrete effect substrate and interaction protein are not clear.We adopt the method for immunoprecipitation to seek interaction factor or the demethylation substrate of ALKBH4 albumen, at first build the pEGFP-C1b-ALKBH4 plasmid, the primer is as sequence table: forward primer SEQID NO.13 and reverse primer SEQ ID NO.12, amplification obtains ALKBH4 cDNA sequence as sequence table SEQ ID NO.1, it is connected into to plasmid order-checking amplification, expresses people ALKBH4 albumen and see sequence table SEQ ID NO.6.Also build the pEGFP-C1b-ALKBH4H169A-D171A-H254A mutant plasmid simultaneously, point mutation primer used has: H169A-D171A mutant primer SEQ ID NO.18 and two point mutation primers of H254A mutant primer SEQ ID NO.19, this plasmid expression people ALKBH4HDH mutants cDNA is shown in sequence table SEQ IDNO.2, and interpreter ALKBH4HDH mutant protein is shown in sequence table SEQ ID NO.7.Be transfected into 293T cell construction stable cell lines.293T/Flag-GFP and the 293T/Flag-GFP-ALKBH4 stable cell lines cultivated are processed to cell 16 hours with 0.04 μ g/ml nocodazole, it is synchronized to mitotic phase, then lysis is collected to Flag-GFP, Flag-GFP-ALKBH4 albumen and and their mutual protein samples by the method for immunoprecipitation.By two groups of sample degenerative treatments, the SDS-PAGE electrophoresis, with silver-colored transfection reagent box, glue is dyeed, also by the efficiency of immunoblot experiment checking co-immunoprecipitation method, result is as Figure 12 a, shown in b simultaneously, our sample sets is compared with matched group has many specific bands, these bands are cut and deliver to Beijing collection Si Jiayang biotechnology company and do mass spectral analysis, and the detection method of use is the analysis of the online LC-MS/MS second order ms of chromatograph mass spectrum, also detects the methylation state of protein simultaneously.After obtaining analysis result, we therefrom screen with a high credibility and are the albumen relevant to cell division, find the albumen such as actin (actin), non-muscle two type myosins (NM II) from result, wherein the credibility of actin is the highest, and has a monomethylation site and the 84th modify for lysine monomethylation (K84me1).Actin being detected, to have methylated peptide section be 69-YPIEHGIVTNWDDME _mkIWHHTFYNELR-95, karyoplasmic ratio m/z=1153.22, charge number 3, molecular weight is MH+ (Da)=3457.661106, and it is than the large 14.02Da of peptide segment molecule amount that does not normally have to modify, and this molecular size range is just in time the size of a methyl group, the mass spectrometric data software analysis, the search Protein Data Bank learns that this modification that methylates is sitting at the position of actin K84, and result is as Figure 12 c, shown in d.

2, whether checking ALKBH4 interacts with actin

For verifying whether ALKBH4 and actin have interaction, we have built the β of HA label-actin recombiant plasmid, the primer sequence is shown in sequence table: forward primer SEQ ID NO.16 and reverse primer SEQ ID NO.17, amplification obtains β-actin cDNA and sees sequence table SEQ ID NO.4, is connected in the pcDNA3-HA plasmid to express β-actin wild-type protein and see sequence table SEQ ID NO.9.At first whether the ALKBH4 of Study of Exogenous interacts with the β of external source-actin, whether with the agarose gel pearl of anti-Flag label, be co-immunoprecipitation experiment detection Flag-GFP-ALKBH4 interacts with HA-β-actin, result shows that Flag-GFP-ALKBH4 can interact with HA-β-actin, as shown in Figure 13 a.We verify again endogenous ALKBH4 and the interaction between β-actin for next step, use respectively β-actin and ALKBH4 antibody to do the co-immunoprecipitation experiment, the endogenous ALKBH4 albumen of result and β-actin pull down mutually get off, result is as Figure 13 b, shown in c, between this explanation ALKBH4 and actin, there is interaction.

Embodiment five: actin K84 is the critical sites of ALKBH4 and NM II combination.

1, the 84th of acitn the lysine to ALKBH4 in conjunction with playing a key effect

The result of front shows that ALKBH4 albumen and actin interact, and obtain on interactional actin albumen having the site K84me1 that methylates in the mass spectrum result, so we guess that this site that methylates is exactly probably the binding site of ALKBH4.In order to prove our conjecture, we become alanine with the rite-directed mutagenesis test kit of Sratagene company by the 84th lysine mutation of the β-actin of HA-β-actin plasmid is K84A, point mutation primer used is shown in sequence table SEQ ID NO.20, express β-actin K84A mutants cDNA and see sequence table SEQ ID NO.5, translation β-actin K84A mutant protein is shown in sequence table SEQ ID NO.10.With the method detection actin K84A sudden change of same co-immunoprecipitation, whether with ALKBH4, interact.By Flag-GFP-ALKBH4 and the common transfection 293T of HA-β-actin cell, Flag label co-immunoprecipitation found that Flag-GFP-ALKBH4 can get off the HA-of external source wild type β-actin pull down, but HA-β-actin K84A mutant pull down can not be got off, with the endogenous actin of actin antibody test whether can in conjunction with, we find that endogenous actin can be by under Flag-GFP-ALKBH4 pull down in two groups of samples, as shown in Figure 14 a, this explanation actin K84 plays a key effect to the combination of ALKBH4.For this conclusion of sufficient proof, we have and have done reverse co-immunoprecipitation, draw 3 * Flag-ALKBH4 with HA-β-actin, found that HA-β-actin can pull down 3 * Flag-ALKBH4, but HA-is β-and actin K84A mutant but cannot, as shown in Figure 14 b, this two experimental evidences explanation actin K84 be ALKBH4 in conjunction with critical sites.

Actin K84 is the binding site of ALKBH4, and our mass spectrum found that actin K84me1 modifies, ALKBH4 or potential protein demethylase, so we guess that ALKBH4 may be the demethylase of actin K84me1, the iron ion binding site of ALKBH4 has been proved to be to determine the critical sites of its biochemical activity, so if ALKBH4 is the demethylase of actin K84me1, so the enzymatic activity critical sites sudden change of ALKBH4 can be made the binding ability of it and substrate strengthen.Imagining our contrived experiment according to this suddenlys change the HDH domain of ALKBH4 fall, see that whether this sudden change affects the interaction ability between ALKBH4 and actin as our imagination, point mutation primer used has: H169A-D171A mutant primer SEQ ID NO.18 and two point mutation primers of H254A mutant primer SEQ ID NO.19, this plasmid expression people ALKBH4HDH mutants cDNA is shown in sequence table SEQ ID NO.2, and interpreter ALKBH4HDH mutant protein is shown in sequence table SEQ ID NO.7.This mutant and the common transfection 293T of wild type cell are done to the co-immunoprecipitation experiment, found that the binding ability between Flag-GFP-ALKBH4HDH mutant and HA-β-actin has strengthened much than wild type, be that Flag-GFP-ALKBH4HDH pulls down more HA-β-actin, as shown in Figure 14 c, this presentation of results ALKBH4 is probably the demethylase of actinK84me1.

2, ALKBH4 and non-muscle two type myosins (NM II) interact

From the mass spectrometric data result obtain ALKBH4 albumen may and non-muscle two type myosins (NM II) between combination is arranged, so we will be with under the immunoblot experiment checking, between them, whether interaction being arranged, so the co-immunoprecipitation that we just do Flag-GFP-ALKBH4 transfection 293T cell experiment, result shows that ALKBH4 can pull down NM II, as shown in Figure 15 a, this explanation NM II may be also interaction factor or the substrate of ALKBH4.

Interaction between Actin and NM II has been found that for a long time, if ALKBH4 can be by actin K84me1 demethylation, this demethylation certainly has function in cell so, can that actin K84 affect the interaction between NM II? find that by searching document existing scientist did crystal structure analysis, result shows that actin K84 site is positioned at the calmodulin binding domain CaM of NM II, so we guess that the modification of actin K84 or change may affect NM II combination.We adopt anti-HA co-immunoprecipitation method to detect the binding ability of HA-β-actin wild type and K84A mutant and NM II in the 293T cell, the binding ability that experimental result shows HA-β-actin K84A and NM II is weak more a lot of than wild type, as shown in Figure 15 b, this explanation actin K84 is the binding site of NM II.Whether we also detect whether GFP-NM II can be in conjunction with HA-β-actin K84A mutant by reverse co-immunoprecipitation method, result shows that GFP-NM II can not be in conjunction with HA-β-actin K84A mutant, but can be in conjunction with wild type HA-β-actin and endogenous actin, as shown in Figure 15 c, this explanation actin K84 is the crucial binding site of NM II.

Embodiment six: the interaction between ALKBH4 and NM II mediates by actin.

According to above result, we have known that ALKBH4 and actin and NM II interact, interaction between actin and NM II is delivered, we want to understand how these three protein moleculars mutually combine now, that three albumen mutually combine or certain albumen connects two other albumen as intermediate? at first verify whether ALKBH4 can affect the combination of actin and NM II, in the 293T cell, the ALKBH4 gene silencing is fallen, three ALKBH4 siRNA sequences used are shown in sequence table: SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, suppress the ALKBH4 protein expression and see sequence table SEQ ID NO.6, result shows that the disappearance of ALKBH4 can not have influence on the interaction between actin and NM II, as shown in figure 16, this explanation ALKBH4 does not mediate the combination of actin and NM II.Second, whether checking NM II gene can have influence on the interaction between ALKBH4, we fall NM II gene silencing in the 293T cell, three NM II siRNA sequences used are shown in sequence table: SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, result shows that NM II disappearance can not have influence on the interaction between ALKBH4 and actin, and as shown in figure 17, this explanation NM II does not mediate the interaction between ALKBH4 and actin to result yet.This result is also by adding NM II inhibitor B lebbistatin to be verified, when cultivating the 293T cell, add the Blebbistatin that final concentration is 5 μ M to process cell 3 hours, receive cell after drug treating and do the co-immunoprecipitation experiment, result shows that NM II inhibitor is on not impact of the interaction between ALKBH4 and actin, just destroyed the combination between NM II and actin, as shown in figure 18, these two experiment show NM II can not affect the interaction between ALKBH4 and actin.The 3rd, whether checking actin can have influence on the interaction between ALKBH4 and NM II, because so the actin kind is many and low actin polymerization inhibitor cytochalasin D (the Cytochalasin D that selects of gene silencing efficiency, CCD), this medicine can suppress actin and aggregates into fibrous actin(F-actin).Be that 1 μ g/ml CCD is added in the 293T cell culture medium by final concentration, cultivate after 1.5 hours and receive lysis, also will add the CCD of 1 μ g/ml in lysis buffer, back is identical with normal co-immunoprecipitation method.Result shows that the interaction caused between ALKBH4 and NM II and actin adding of actin inhibitor destroys, and as shown in figure 19, this explanation actin is the interactional middle element between ALKBH4 and NM II.According to above our inference ALKBH4 as a result and NM II, be combined on the actin fiber, the actin fiber has mediated the interaction between ALKBH4 and NM II simultaneously.

Embodiment seven: ALKBH4 is the demethylase of actin K84me1.

It is existing that we know that ALKBH4 and actin mutually combine as a result, actin K84 be ALKBH4 in conjunction with critical sites, ALKBH4 enzymatic activity domain inactivation causes strengthening with the combination of actin, and in the mass spectrum result, actin K84me1 has a monomethylation site, all these evidences show that ALKBH4 is probably the demethylase of actin K84me1.In order to verify that at first this conjecture has prepared the antibody for actinK84me1, look for the antibody of U.S. New England Peptide company preparation for actin K84me1, they obtain antiserum with the upper 77-TNWDDMEmKIWHHTFY-91 of the actin peptide section immune rabbit of modifying that methylates, with Protein A antibody purification, then with the methylate antibody for actin K84me1 of chromatographic column purification specificity of polypeptide of coupling.

Checking antibody: we will methylate modify containing the K84me1 polypeptide and do not methylate the K84 polypeptide point modified to nitrocellulose filter, and hybridize with this antibody, result shows the polypeptide that the identification of actin K84me1 antibody specificity methylates and modifies, the polypeptide that nonrecognition does not methylate and modifies, identical with Ponceau S dyeing proof applied sample amount, result is as shown in Figure 20 a, we also verify the specificity of antibody with heterogenous expression HA-β-actin wild type and K84A transfection with mutant 293T cell simultaneously, result proves that this antibody can not identify HA-β-actin K84A but can identify wild type, as shown in Figure 20 b, the antibody of these two result proof anti-actin K84me1 has very special identification ability, can be used for verifying that the expression of actin K84me 1 changes.

Verify that by immunofluorescence technique actin K84me1 is in intracellular location, we hybridize as label and the actinK84me1 of retraction ring and middle body jointly with a-tubulin, and identify two kinds of primary antibodies with two kind of two anti-hybridization of coupling Cy3 and FITC molecule, result shows that actin K84me1 is positioned on retraction ring and middle body, as shown in figure 21.

Checking ALKBH4 is actin K84me1 demethylase: we first verify whether ALKBH4 is the demethylase of actin K84me1 in vivo, at first, the ALKBH4 gene is reticent in the U2OS cell, three ALKBH4 siRNA sequences used are shown in sequence table: SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, suppressing endogenous ALKBH4 Protein S EQ ID NO.6 expresses, the ALKBH4 gene silencing detected and cause that actin K84me1 expression increases considerably, as shown in Figure 21 a, cross expression external source 3 * Flag-ALKBH4 causes actin K84me1 expression to reduce in a large number simultaneously, excessively expressing 3 * Flag-ALKBH4HDH point mutation in the U2OS cell causes actin K84me1 expression to increase, as shown in Figure 21 b, these two presentation of results ALKBH4 wild-type proteins in vivo can be by the actinK84me1 demethylation, and the HDH mutant does not have this activity.Crossing the insensitive 3 * Flag-ALKBH4 of expression ALKBH4 siRNA in the U2OS cell can make actinK84me1 get back to normal level simultaneously, and 3 * Flag-ALKBH4HDH of enzymatic activity domain sudden change can not, as shown in Figure 21 c, this explanation 3 * Flag-ALKBH4 wild type has the demethylation activity, and the HDH mutant does not have activity.The co-immunoprecipitation experiment is found in addition, ALKBH4 and NM II can pull down actin, but the actin majority that ALKBH4 is left behind is methylated, and the actin that NM II pulls down does not methylate to modify, as shown in Figure 21 d, this explanation actin K84me1 disturbs the combination of NM II, only has ALKBH4 by above after actin K84me1 demethylation, NM II can be combined in, and the demethylation activity of ALKBH4 is the key factor that NM II slides along the actin fiber.These results suggest that ALKBH4 albumen is the demethylase of actin K84me1 in vivo, ALKBH4 provides binding site by actin K84me1 demethylation for NM II is combined on actin, also for NM II, on the actin fiber, slide condition is provided, and ALKBH4 is positioned on retraction ring, the major impetus source that retraction ring shrinks is exactly that the slip effect of NM II on the actin fiber produces, therefore the decisive molecule that ALKBH4 thing NM II slides on actin, it is determining that can retraction ring shrink cytokinesis

Embodiment eight: ALKBH4 regulates and controls cytokinesis, and the ALKBH4 disappearance causes the cytokinesis failure.

The interaction protein Pseudobulbus Bletillae (Rhizoma Bletillae) demethylation substrate of the existing known ALKBH4 of result, but we also do not know which type of cytobiology is its these mechanism can cause change, in order to verify that controlling retraction ring about ALKBH4 in a upper embodiment shrinks the guess that determines cytokinesis, we adopt immunofluorescence, Flow Cytometry to go checking.Research in front finds that ALKBH4 is positioned on retraction ring, middle body and centrosome, so we concentrate on the emphasis of research on fissional phenomenon.At first we are by ALKBH4 gene silencing in the MRC5 cell, three ALKBH4 siRNA sequences used are shown in sequence table: SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, suppressing endogenous ALKBH4 Protein S EQ IDNO.6 expresses, in detecting with the label AuroraB of middle body and Plk1, body structure changes, find in the MRC5 cell that after the ALKBH4 silence, obvious change has all occurred in the celluar localization of Aurora B and Plk1, as Figure 23 a, shown in b, middle body structure abnormal after this explanation ALKBH4 gene silencing.ALKBH4 gene silencing rear center body over-replicate detected with centrosome label γ-tubulin, compare ALKBH4 gene silencing rear center body over-replicate with matched group and surpass more than five times, as shown in Figure 23 c.We detect ALKBH4 gene silencing after-contraction ring structure with the label NM II of retraction ring and anillin and also occur extremely, and main manifestations is that the anillin location extremely can't form retraction ring and form the contracting ring in a plurality of positions of cell, as Figure 23 d, shown in e.After this explanation ALKBH4 gene silencing, cell division is the cytokinesis abnormal especially.

Retraction ring and middle body play a key effect in cytokinesis, if they cytokinesis time lengthening or cytokinesis failure occur extremely may causing, whether the splitting time of at first verifying cell extends, if the most obvious feature of cell division time lengthening is that division cells quantity increases, therefore dye DNA with propidium iodide (PI), more whether increase with G2/M phase cell after ALKBH4 gene silencing in flow cytometer detection MRC5 cell.After experimental result shows the ALKBH4 gene silencing, the G2/M cell obviously increases, and sees that from statistical result the ALKBH4 gene silencing causes G2/M phase cell to increase by one times, as shown in Figure 24 a.In order further to determine whether M phase cell increases, and we carry out labelling M phase cell with the antibody of the specific marker thing histone H 3-S10p of anti-M phase cell, in result demonstration MRC5 cell after the ALKBH4 gene silencing M phase cell double above, as shown in Figure 24 b.Also found that expression 3 * Flag-ALKBH4HDH and HA-β-actin K84A mutant also caused that the MRC5 cell M phase significantly increases simultaneously, as shown in figure 25, show that 3 * Flag-ALKBH4HDH compares afunction with HA-β-actin K84A mutant with wild type, the overexpression mutant causes endogenous wild-type activity to suppress.This explanation ALKBH4 gene silencing can cause the retardance of cell cycle, especially in division stage, front found that the ALKBH4 gene silencing affects retraction ring and forms and middle body structure, these presentation of results ALKBH4 gene delection causes cell division to be arrested in the cytokinesis phase, so ALKBH4 plays a key effect in the regulation and control cytokinesis.Since the ALKBH4 gene silencing can cause that cell block is in the cytokinesis phase, how can the result of back be so? be interfered and can cause the cytokinesis failure according to bibliographical information cytokinesis process, finally can cause apoptosis or form apocyte.Therefore we imagine the ALKBH4 gene silencing and whether have same consequence, detect the label with α-tubulin as cytoskeleton by the method for immunofluorescence.Experimental result shows in the MRC5 cell to produce after the ALKBH4 gene silencing a large amount of apocytes, and the quantity that apocyte produces is more than five times of matched group, as shown in Figure 26 a.We are also with apoptosis test kit labelling and use the apoptosis situation after flow cytometer detects the ALKBH4 gene silencing, result shows in the MRC5 cell after the ALKBH4 gene silencing that a large amount of apoptosis have occurred cell, see that from statistical result ALKBH4 gene silencing group is three times of matched group, as shown in Figure 26 b.The phenomenon of the cytokinesis failure that above ALKBH4 gene silencing causes is verified in the living cells imaging experiment, we use the Laser Scanning Confocal Microscope of come card company to observe the process that in the MRC5 cell, after the ALKBH4 gene silencing, cell cytoplasm divides, after found that the ALKBH4 gene silencing, two final results have appearred in cytokinesis: the one, and cytokinesis does not complete two daughter cells and merges again that this is corresponding with the apocyte phenomenon, another phenomenon is that cytokinesis continues to complete last apoptosis, as shown in Figure 26 c.Retraction ring that the ALKBH4 gene silencing produces location is abnormal, M phase cytosis and apocyte can be repaired by the insensitive Flag-GFP-ALKBH4 of heterogenous expression siRNAture, but Flag-GFP-ALKBH4 HDH mutant can not, as shown in figure 27.This makes us reason out the action model of ALKBH4 albumen in the cytokinesis process, in the cytokinesis stage, ALKBH4 and NM II are combined on the actin fiber simultaneously, ALKBH4 is combined on the 84th, the place ahead methylated actin of lysine of NM II glide direction, and the actin of NM II combination does not methylate to modify, ALKBH4 albumen in NM II the place ahead constantly by actin K84me1 demethylation, this provides condition along the actin fiber to front slide for NM II, make and take actin fiber and NM II and can produce contractility as basic retraction ring, impel cytokinesis to complete, as shown in figure 28.

Since the ALKBH4 gene silencing can cause the cytokinesis failure in the normal MRC5 cell of people, be in tumor or cancer cell, how which type of reaction can be so? in order to identify the reaction of ALKBH4 gene silencing in tumor cell, we have selected several tumor cells, as human osteosarcoma cell U2OS, oral squamous cell carcinoma cell OSC20 and ovarian cancer cell TOC2.At first, reticent ALKBH4 gene in the U2OS cell, dye DNA with propidium iodide (PI), after detecting the ALKBH4 gene silencing with flow cytometer again, whether G2/M phase cell increases, result shows in the U2OS cell after the ALKBH4 gene silencing that the G2/M cell obviously increases, see that from statistical result the ALKBH4 gene silencing causes G2/M phase cell to increase by one times, as shown in Figure 29 a.In order further to determine whether M phase cell increases, carry out labelling M phase cell with the antibody of the specific marker thing histone H 3-S10p of anti-M phase cell, in result demonstration U2OS cell after the ALKBH4 gene silencing M phase cell double, as shown in Figure 29 b.We are also with apoptosis test kit labelling and use the apoptosis situation after flow cytometer detects the ALKBH4 gene silencing, and result shows in the U2OS cell after the ALKBH4 gene silencing that a large amount of apoptosis have occurred cell, as shown in Figure 29 c.Also produce a large amount of apoptosis after the ALKBH4 gene silencing equally in oral squamous cell carcinoma cell OSC20 and ovarian cancer cell TOC2 cell, as shown in figure 30.These presentation of results ALKBH4 gene silencing can cause tumor and cancer cell cytokinesis failure finally to cause a large amount of apoptosis.

In sum, all antibody of the human actin actin K84me1 that the 84th lysine monomethylation of all antisense RNA sequences, people of the human actin actin K84me1 that the 84th lysine monomethylation of human actin actin K84me1 siRNA sequence, people that the 84th lysine monomethylation of people modified modified modified all can prevent or treat cancer, and described cancer comprises human osteosarcoma, oral squamous cell carcinoma and ovarian cancer etc.Embodiment nine: ALKBH4 knock out mice death during embryonic period, ALKBH4 knock out mice cell cytoplasm divides and unsuccessfully produces apocyte and follow apoptosis

In order to study ALKBH4 gene function in animal body, we build knock out mice, mice ALKBH4 gene has three exons, wherein 2, the enzymatic activity key structure territory that 3 exons have comprised the ALKBH4 gene, as shown in Figure 31 a, so these two key structure territory knock-out mice ALKBH4 albumen can not be expressed and produced the ALKBH4 knock out mice.Traditional ALKBH4 knock out mice is lethal the period of embryo, and we are by ALKBH4 ^+/-there is no ALKBH4 in the mice of hybridization birth ^-/-mouse genotypes, birth mice ratio meets Mendel's law 1 (+/+): 2 (+/-): 0 (1,-/-), as shown in Figure 31 b, this explanation ALKBH4 gene is extremely important at Embryonic Stages.This result and actin gene knockout come to the same thing, and now existing bibliographical information is lethal by the mice of actin gene knockout the period of embryo.

In order to obtain the ALKBH4 knock out mice of survival, but we build the ALKBH4 knock out mice of induction type, equally by 2, 3 exons knock out, while building gene knockout carrier 2, 3 exon both sides add the LoxP site of two recombinase Cre identifications, as shown in Figure 32 a, PCR primer used is shown in sequence table forward primer SEQ ID NO.21 and reverse primer SEQ ID NO.22 amplification leading portion homologous recombination exons 1 sequence, the exon 2 that forward primer SEQ ID NO.23 and reverse primer SEQ ID NO.24 amplification gene knock out, 3 sequences, forward primer SEQ IDNO.25 and reverse primer SEQ ID NO.26 amplification exon 3 back homologous recombination sequence, segmentation amplification mice ALKBH4 genomic dna sequence like this, see sequence table SEQ ID NO.3, this fragment gene group sequence obtains mice ALKBH4 albumen after transcription and translation, see sequence table SEQ ID NO.8, knock out mice can not be expressed this section protein sequence.The ALKBH4 gene knockout carrier electricity built is forwarded in embryonic stem cell, and southern blotting experiment detects gene knockout carrier and is incorporated in embryonic stem cell, as shown in Figure 32 b.The embryonic stem cell microinjection is brought out to ALKBH4 to C57BL/6N mice ascus embryo ^+/Lmouse genotypes, more this mouse genotypes hybridization is obtained to ALKBH4 ^l/Lmouse genotypes.Is ALKBH4 by this mouse genotypes and the mice copulation of isozygotying that contains the Cre zymophore to genotype ^+/L/Cremice, feed mice with TAM and can produce ALKBH4 ^+/-/Cregenotypic mice, then verify this heterozygote knock out mice with RT-PCR, as shown in Figure 32 c.ALKBH4 ^+/-/Cregenotypic mice copulation obtains ALKBH4 ^-/-/Cre, use the RT-PCR method validation, as shown in Figure 32 d.

Get ALKBH4 ^-/-/Cre3.5 days embryos of mouse genotypes separate the MEF fibroblast, add 4-OHT and induce the ALKBH4 gene knockout, and detect the expression of ALKBH4 and actin K84me1 with immunofluorescence and immunoblot experiment, result shows that ALKBH4 gene knockout efficiency is very high, in the MEF of ALKBH4 gene knockout cell, the expression of actin K84me1 obviously raises, consistent with ALKBH4 gene silencing result, as shown in figure 33.Immunofluorescence experiment also finds that the MEF cell after the ALKBH4 gene knockout produces a large amount of apocytes, and the phenomenon of over-replicate has appearred in centrosome, as shown in figure 34.We also find after the ALKBH4 gene knockout that MEF cell proliferation and transfer ability die down, ALKBH4 gene knockout cell and cellular control unit are marked to a white space, after 20 hours, cellular control unit fills up this sheet white space, and the white space of ALKBH4 gene knockout group size does not change, this explanation ALKBH4 also plays very important effect in cell proliferation and transition process, as shown in figure 35.

Sequence table

<110 > Beijing Institute of Gene Science, Chinese Academy of Sciences

<120 > a kind of gene and encoding proteins thereof promote cell cytoplasm division, cell proliferation and apply in medicament research and development

<160>32

<170>

<210>1

<211>909

<212>DNA

<213 > people (Homo Sapiens)

<220>Homo?sapiens?ALKBH4?cDNA?sequence

<400>1

atggcggcgg?ctgccgccga?gacccccgaa?gtccttcggg?aatgcggttg?caagggcatc 60

cggacctgtc?tgatctgcga?gcggcagcgc?ggcagtgacc?cgccctggga?gctgccccca 120

gcgaaaacat?accgtttcat?ttactgctcc?gacaccggct?gggccgtggg?cacagaggag 180

tctgactttg?agggctgggc?cttccccttc?ccaggagtga?tgctgatcga?ggactttgtg 240

acccgggagg?aagaagccga?gttggtgcgg?ctcatggacc?gtgacccctg?gaagctctcc 300

cagtctggac?ggaggaagca?ggactatggc?cccaaagtca?actttcggaa?acagaagcta 360

aagaccgagg?gcttctgcgg?cctccccagc?ttcagccggg?aggtggtgcg?gaggatgggc 420

ctctacccgg?ggctggaggg?cttccggccc?gtcgagcagt?gcaacctgga?ctactgcccc 480

gagcggggct?ctgccattga?cccccacctg?gacgacgcct?ggctgtgggg?ggagcggctg 540

gtcagcctca?acctcctgtc?ccccaccgtg?ctgtccatgt?gtcgggaggc?gcccgggagc 600

ctgctcctct?gctcggcccc?gtcggctgcc?ccggaggcct?tggtggacag?cgtgatagca 660

cccagccggt?cggtgctatg?ccaggaggtg?gaggtggcca?tccccttacc?cgcccgctcc 720

ctgctggtcc?tcaccggggc?ggcacggcac?cagtggaagc?atgccatcca?ccgcagacac 780

atcgaggccc?gccgcgtctg?cgtcactttc?cgggagctgt?cggctgagtt?tggccctgga 840

gggaggcagc?aagagctggg?ccaggaactg?ctgcggatcg?ccctctcctt?ccagggaaga 900

cccgtgtga 909

<210>2

<211>909

<212>DNA

<213 > people (Homo Sapiens)

<220>Homo?sapiens?ALKBH4?H169A-D171A-H254A?mutation?cDNA?sequence

<400>2

atggcggcgg?ctgccgccga?gacccccgaa?gtccttcggg?aatgcggttg?caagggcatc 60

cggacctgtc?tgatctgcga?gcggcagcgc?ggcagtgacc?cgccctggga?gctgccccca 120

gcgaaaacat?accgtttcat?ttactgctcc?gacaccggct?gggccgtggg?cacagaggag 180

tctgactttg?agggctgggc?cttccccttc?ccaggagtga?tgctgatcga?ggactttgtg 240

acccgggagg?aagaagccga?gttggtgcgg?ctcatggacc?gtgacccctg?gaagctctcc 300

cagtctggac?ggaggaagca?ggactatggc?cccaaagtca?actttcggaa?acagaagcta 360

aagaccgagg?gcttctgcgg?cctccccagc?ttcagccggg?aggtggtgcg?gaggatgggc 420

ctctacccgg?ggctggaggg?cttccggccc?gtcgagcagt?gcaacctgga?ctactgcccc 480

gagcggggct?ctgccattga?cccc gccctg?g ccgacgcct?ggctgtgggg?ggagcggctg 540

gtcagcctca?acctcctgtc?ccccaccgtg?ctgtccatgt?gtcgggaggc?gcccgggagc 600

ctgctcctct?gctcggcccc?gtcggctgcc?ccggaggcct?tggtggacag?cgtgatagca 660

cccagccggt?cggtgctatg?ccaggaggtg?gaggtggcca?tccccttacc?cgcccgctcc 720

ctgctggtcc?tcaccggggc?ggcacggcac?cagtggaag g? ctgccatcca?ccgcagacac 780

atcgaggccc?gccgcgtctg?cgtcactttc?cgggagctgt?cggctgagtt?tggccctgga 840

gggaggcagc?aagagctggg?ccaggaactg?ctgcggatcg?ccctctcctt?ccagggaaga 900

cccgtgtga 909

<210>3

<211>6635

<212>DNA

<213 > Mus (Mus Sapiens)

<220>Mouse?mALKBH4?genome?DNA?sequence

<400>3

cccgttccgt?gctacacagg?atccctcaac?atcccttagt?atctcatcag?tctttccact 60

ccgcgcaagg?ctaagggcgt?tcgctccaag?tttatctgac?gccgggcgtt?ggctgccaat 120

ctcccacatc?tccgtttggc?gcgagattcg?actggggctc?gaatcccgga?taacagaata 180

acccgggcag?gaggaggaga?cggaggggtc?tgagccttgc?attccgccca?tcaaaccacc 240

ctggcgggga?agaaccccac?gccccggatc?cgcttcaacc?ccacatgctc?ttacttcaaa 300

ctctgaatct?tccccagctt?atccgtcttg?ggtcgcccac?gttgcaggag?cagttgcggc 360

gtgagaggag?ccatgggtgg?ccgagggctc?aaccgaaccc?gcgcctccac?gaagtccggt 420

cgctaaggcc?cgctgccggg?gagcccagga?ggtggcgcac?aacaaaccct?ttgcgagtcc 480

gttgcgaccg?gaaggaagta?gaacgccagc?cttacgcccc?acctcctgct?tctgcgatga 540

ctacaactcc?cagcggcccc?tgcgtctcgc?gcctgcgctc?ttaactgatc?ggaggacgcg 600

atggcggcgg?cggctgaagt?ttctcttttg?caggagtgcg?gctgtaaggg?catccggacc 660

tgtcttatct?gcgagagaca?acgccacagg?gacccgccct?ggcagatctg?ccttcaggta 720

cggactagga?aaagagtcat?agccagaaaa?tctattcatc?ttaatgggaa?actaaggccc 780

agcttgcctg?taaaacagcc?aaggaccaaa?gcctcgcggt?aggtgagttg?tttggagcgc 840

ttatatcttc?aatattcaca?aaacccaaaa?ggtggaccaa?gtgccatcgg?ctgattaatg 900

gaagggcggg?gaaatgctgg?tgcacgccga?gatctctgca?tttgaaaggc?agagaggctg 960

gagaacggac?ctcagccgcc?cagcaggttt?taggagttac?cttggcagta?gggaagcggc?1020

tgtcacacat?catctctgtg?actttggggg?gtgaggggtg?ggggtggggg?gcagtggtaa?1080

ataacttgga?ctgaacgggt?ctggttttga?gtgagcttcc?ggagcacaag?ggagactgca?1140

tggaaatggt?agggagtgct?gaacactagg?ccgcctgtat?tagacaggga?atgagcgact?1200

taagacagca?caggcagccg?ggcagtggtg?gcgcatgcct?ttaatcccag?cactcgggag?1260

acaagaggca?ggtggaattc?cgagttcgag?gccagcctgg?tctacagagt?gagttccagg?1320

acagccagga?ctacactaca?gagaaacctg?tctccaaaac?aggctttgat?ttgggggaaa?1380

tttttttggg?aaaacaaacc?tgtatgaatc?agctttacag?ccgggcagtg?gtggtgcatg?1440

ccattagact?tagcacttgg?gatgcagagg?caggcagatc?tctgtgagtc?ccaggctagc?1500

ctgatctaca?tagggagttc?caggaaagcc?catcttgaaa?aacaaaaaca?aatctgtctt?1560

acaagatact?aataggatca?ggagtcaggt?aggccaggaa?gaatgttaaa?atgtttccct?1620

tctagctctc?tgttgaccag?gctaacacct?tccatcaaaa?tgggcaatgg?atggatgaat?1680

gaatgaatga?atgaatgaaa?aataacttca?gagcatttgt?atggccctgg?cagtgctgta?1740

gcaagttgct?gggatgcata?gccaggggaa?tgcttgagta?gaggcctgag?ataggagctg?1800

tgggtggctg?aggcgacaga?cacttaaaga?gatcaaaggg?gcctgagcca?agttttttgt?1860

tttagttatt?ttctttagtt?agaggcccca?gggcgccccc?tccataagct?aagattgcaa?1920

gcatatgtca?ctatgtctgg?atcatggcag?cattcagagt?gaccaaacac?ccaagtgttt?1980

attatatcaa?ctggtgaact?gaagagtggg?cagtgttggt?gctggcttga?gggaggtcaa?2040

agatatctac?atagcaagtt?tcaaaccaga?ctaaataaaa?gcctgtctcg?acaggaaaga?2100

aagaggcaaa?ccataaacta?ataagagatt?tttaaaaatc?tcggatatcc?atgcagtaga?2160

tataacctta?gaaaggaagg?aaattctgac?gcgagttcca?tatggattca?ccttgaagat?2220

gtgctgctaa?aagaaggaag?ccagtctcta?gctggaggcg?tggtctggag?atagtgcacc?2280

tgcctagcac?cgcccaggct?ggagagatgg?ctctacactt?aagaacactg?actgcttcgg?2340

gcgtgctggc?gcaggccttt?aatcccagca?ctcgggaggc?agaggcaggc?ggatttctga?2400

gttcgaggcc?agcctggtct?acagagtgag?ttccaggaca?gccagggcta?cacagaaaaa?2460

ccctgtctca?gaaaaaaaaa?accaaagacc?taagtttgcc?tcccaacacc?caagtcactg?2520

gcgccctctt?ctggccttgg?agggcacatg?tgcatgccac?acacagattc?aaacatagaa?2580

agaaagaact?gccctagtga?ggggcttggg?atggacctct?gtggtagagt?gcttacctaa?2640

catgctaagg?cctggctcat?gtagcccagg?ctggccttat?tcactatata?gtgaaagatg?2700

accatgaact?cttaatggtc?ctgaagacca?ggattacaga?cctctgccgc?cacacaggtt?2760

tttttgcagt?gctggggaag?gaacccatgg?cttcatgcat?gtcgggcagg?cctataccat?2820

ctgagctaca?gccacagatt?tatgatgccc?tacattttat?gtacattggt?gttttgcctg?2880

catgtgtgtc?tgtgtgaggg?tgttggatct?cctggaactg?aagttaatgg?acagtcgtga?2940

gctctcccgg?ttggttgtct?ggaagagcgg?ccagtgcctt?tattcgccga?gccttctctc?3000

cagccctaag?atggcgtttc?tactactgca?tctcttagtc?tcagaggagg?caggagcagg?3060

gctgaggaag?acacacatct?gtggcttcca?ataagcattg?gccagcccct?ggccacccag?3120

accacagcct?tacggttagc?cctgcttctc?agactcctgg?tgcaggctcc?tgtcctttat?3180

ctttttgctc?ctgaggggtc?ggtgatagtc?acagtgccaa?aggaatagtg?cccacatccc?3240

ccagcctcag?gccaggtcct?cagagaagaa?aaacacaaat?gagagaatga?gaatcccatg?3300

ccctgaggcc?tgaggccagg?ttcaggctgc?atcgcccctg?cgactccagg?ctcctctcca?3360

agttgcttta?gttccaagaa?gagcatcgga?atcccagagg?ctcctggtgg?gtcccgggca?3420

catgtggcta?ttgtgggtct?ttgctggttc?cagggcactt?ctgtttgggg?tgtatgcgtg?3480

tacaggtgta?tgtgtataca?ggtagagggc?agaggataac?ccaggtgccc?accttggtgt?3540

tttgagagag?tctcactggc?ctggaatttt?ctaagaggcc?aggctagctt?gcccttggtc?3600

tgacctttgc?ctccctcccc?agctctagga?tacaggcatc?catgaccaca?cccagcttat?3660

tacatttggg?ttttagggga?tggagctcag?atctctgtgc?ttcttgcagg?ttaagcacgt?3720

cagcagctca?cccactctgc?cccatctctg?tcttttacag?aaaaaatgct?gtttcctcta?3780

ctgcccagac?acaggctggg?ccgcaggggc?cgagggctct?gacttggagg?gttgggcatt?3840

ccccttccca?ggagtgacac?tgatacagga?ctttgtgacc?ccagaggaag?aagctgagat?3900

ggtgcgtctg?atggactgcg?acccctggaa?gctctcacag?tctgggagga?agaagcaggt?3960

acggtggcct?gagctgagcc?gaggtatggt?ccctttgcca?gtggccccgg?ctacctcctg?4020

tagccacacc?ctagggatct?ttggtcacag?tgggtgtgga?cttcctcatc?aagcgtggct?4080

gccgcgtctc?tgtgcggctg?tctctgcggc?tgtctcgttg?gtgaggggat?gccaagctct?4140

aggttggatt?tctatcctgt?gtgctgctag?aggctgccgg?gcatcacccg?tttctattgt?4200

gactctcact?cctcacctgt?atgtctcacc?ttctatcttt?tcccttcctc?gttatctctc?4260

ccttgccaca?atgtagtcag?tgccttagta?gcttcaaagg?tgcccctcac?cctttgctcc?4320

tggcaccata?gtgctgggtt?agagttctgg?cgacatcagc?cagttggggg?tcccttcaag?4380

cttcgtcctg?tgctgggaca?gccccagcca?cccactctgc?ccctgacctc?tgaacagatg?4440

gagcagtcct?cctacctagg?ctttctgggt?cctgggattt?catgcacaca?ccagccatgc?4500

ccagtttgtg?gttgccatgg?cttaagctgg?gccacttctc?cttcctgtct?ccactgtagg?4560

actatggccc?taactgcttc?tccttcctgt?ctccactgca?ggactatggc?cctaaagtta?4620

attttcggaa?gcagaagctg?aagatggcag?gcttccaggg?tctccctggc?tttagccaga?4680

aggtagtcca?gagaatgggc?ctttacccgg?ggctggagga?cttccagcct?gtggaacagt?4740

gcaatctgga?ctacagccct?gagcggggct?ccgccatcga?cccccacttg?gacgacgcct?4800

ggctgtgggg?cgagcggctg?gtgagcctca?acctcctgtc?agccacagtg?gtgtccatgt?4860

ctccagaggc?ccctggcagc?ctgcttctct?gctcagcccc?ctccgtcaga?cctgatgctt?4920

ttgaggacag?ccttgtggct?cctagcaggt?ctgtcccctg?ccaggaggtg?gaggtggcca?4980

tcacagtacc?ccgccgctcc?ttgctggtgc?ttacaggggc?cgcgaggcac?cagtggacac?5040

acgccatcca?ccgcagacac?atcaaggccc?gccgtgtgtg?cgccaccttc?agggagctgt?5100

ccagtgagtt?cctgcctgga?gggaagcagc?aagaactggg?ccaagaactg?ctgcaggctg?5160

cactctcgtt?ccaggggaga?ccggtgtgat?gcttcccagg?gcccaggagc?tgtcactggc?5220

ctggccaaaa?gcttggctcc?agcacaacca?tcggaggagc?taacactgag?gtctccccac?5280

agcagtagat?tcgtggggtt?tttttgagac?aaggtctcat?gtagcccagg?ctagcctcca?5340

attaattgtg?tagctaagga?tgaccttgga?ctcttgcctt?ttctacctcc?tcttcccaag?5400

ctctggcttg?attattttgt?tttgtttctc?tttttgggac?agggtctccg?gcctagactg?5460

gcctcagact?ctgtttccag?ccaaggatga?ctttgaactc?cggatcctcc?tgcctctacc?5520

tcttgaatgc?tgggattgcc?agcgtgcacc?atcatatgca?atttttgtct?gtgctgggat?5580

ggcagcaagc?gttctgccag?ctgagctacc?ccacccccct?agcctgttgg?gggtttttga?5640

gacaaggcct?cacatgggct?agactggcct?ggaactcccc?atcctcctgc?ctcagcctcc?5700

cagtcgctga?ggtgacagtg?tactggtgag?gttttaggag?gagccatctg?acccttgacg?5760

tcatgagcat?cgtatgtcct?gcccggagct?ggttctgctg?ggcctgggct?gcggccattc?5820

ctcagctcac?tgagtgtcat?atgtggaggg?attgagtctt?ttagaacttc?agtacttggg?5880

acgagtaagg?tggctcagta?gctaaaggtg?ctgccaccaa?gcctgatgtt?cagtctctgc?5940

aatccacgtg?gtaaaaggag?tgtcccctga?tctccacacg?cacatcaccc?attttgataa?6000

aattcataat?aaaagaaaaa?ccttggtctc?atgtgaaatg?aatttctcct?tttcccatcc?6060

ctgtgtgaag?tagttgtagt?tattttgttt?tgttttaaaa?cagggtctca?ctttgtagcc?6120

ttggctgtct?tggaactcgc?tttatagacc?aggctggccc?ttaactcata?aagatctcta?6180

cctctgccgc?tgagtaagtg?ccgggattaa?agatatgcac?caatccacag?agcccaactc?6240

tttcttttct?tttctttttt?tttttttttt?tgagataagc?ttcccatttg?tagtacaggc?6300

tggcctcaaa?atttgaccag?tcaggaccca?aggattaaac?ttagttttaa?aaattacatt?6360

gacttagtta?tttagctatg?gaagcaggca?cagctctgtg?tatggaggac?agaagaccat?6420

gcaggccaag?gtggaaggtg?gagttgtgtg?gctggcctgc?atgacagcct?tgtacctcca?6480

ggaaagggtc?acagcctcgg?ggtttgtgat?accaagtttt?gggaggcttg?tgtttctgtg?6540

tgcagattgc?agacttatgt?gggtgcagtg?cacttgctct?gtgcatgcat?cagggggcca?6600

gctgcaatcg?catgcacgtg?gtatccgtga?ggtca?6635

<210>4

<211>1128

<212>DNA

<213 > people (Homo Sapiens)

<220>Homo?sapiens?β-actin?cDNA?sequence

<400>4

atggatgatg?atatcgccgc?gctcgtcgtc?gacaacggct?ccggcatgtg?caaggccggc 60

ttcgcgggcg?acgatgcccc?ccgggccgtc?ttcccctcca?tcgtggggcg?ccccaggcac 120

cagggcgtga?tggtgggcat?gggtcagaag?gattcctatg?tgggcgacga?ggcccagagc 180

aagagaggca?tcctcaccct?gaagtacccc?atcgagcacg?gcatcgtcac?caactgggac 240

gacatggaga?aaatctggca?ccacaccttc?tacaatgagc?tgcgtgtggc?tcccgaggag 300

caccccgtgc?tgctgaccga?ggcccccctg?aaccccaagg?ccaaccgcga?gaagatgacc 360

cagatcatgt?ttgagacctt?caacacccca?gccatgtacg?ttgctatcca?ggctgtgcta 420

tccctgtacg?cctctggccg?taccactggc?atcgtgatgg?actccggtga?cggggtcacc 480

cacactgtgc?ccatctacga?ggggtatgcc?ctcccccatg?ccatcctgcg?tctggacctg 540

gctggccggg?acctgactga?ctacctcatg?aagatcctca?ccgagcgcgg?ctacagcttc 600

accaccacgg?ccgagcggga?aatcgtgcgt?gacattaagg?agaagctgtg?ctacgtcgcc 660

ctggacttcg?agcaagagat?ggccacggct?gcttccagct?cctccctgga?gaagagctac 720

gagctgcctg?acggccaggt?catcaccatt?ggcaatgagc?ggttccgctg?ccctgaggca 780

ctcttccagc?cttccttcct?gggcatggag?tcctgtggca?tccacgaaac?taccttcaac 840

tccatcatga?agtgtgacgt?ggacatccgc?aaagacctgt?acgccaacac?agtgctgtct 900

ggcggcacca?ccatgtaccc?tggcattgcc?gacaggatgc?agaaggagat?cactgccctg 960

gcacccagca?caatgaagat?caagatcatt?gctcctcctg?agcgcaagta?ctccgtgtgg?1020

atcggcggct?ccatcctggc?ctcgctgtcc?accttccagc?agatgtggat?cagcaagcag?1080

gagtatgacg?agtccggccc?ctccatcgtc?caccgcaaat?gcttctag 1128

<210>5

<211>1128

<212>DNA

<213 > people (Homo Sapiens)

<220>Homo?sapiens?β-actin?K84A?mutation?cDNA?sequence

<400>5

atggatgatg?atatcgccgc?gctcgtcgtc?gacaacggct?ccggcatgtg?caaggccggc 60

ttcgcgggcg?acgatgcccc?ccgggccgtc?ttcccctcca?tcgtggggcg?ccccaggcac 120

cagggcgtga?tggtgggcat?gggtcagaag?gattcctatg?tgggcgacga?ggcccagagc 180

aagagaggca?tcctcaccct?gaagtacccc?atcgagcacg?gcatcgtcac?caactgggac 240

gacatggag g? caatctggca?ccacaccttc?tacaatgagc?tgcgtgtggc?tcccgaggag 300

caccccgtgc?tgctgaccga?ggcccccctg?aaccccaagg?ccaaccgcga?gaagatgacc 360

cagatcatgt?ttgagacctt?caacacccca?gccatgtacg?ttgctatcca?ggctgtgcta 420

tccctgtacg?cctctggccg?taccactggc?atcgtgatgg?actccggtga?cggggtcacc 480

cacactgtgc?ccatctacga?ggggtatgcc?ctcccccatg?ccatcctgcg?tctggacctg 540

gctggccggg?acctgactga?ctacctcatg?aagatcctca?ccgagcgcgg?ctacagcttc 600

accaccacgg?ccgagcggga?aatcgtgcgt?gacattaagg?agaagctgtg?ctacgtcgcc 660

ctggacttcg?agcaagagat?ggccacggct?gcttccagct?cctccctgga?gaagagctac 720

gagctgcctg?acggccaggt?catcaccatt?ggcaatgagc?ggttccgctg?ccctgaggca 780

ctcttccagc?cttccttcct?gggcatggag?tcctgtggca?tccacgaaac?taccttcaac 840

tccatcatga?agtgtgacgt?ggacatccgc?aaagacctgt?acgccaacac?agtgctgtct 900

ggcggcacca?ccatgtaccc?tggcattgcc?gacaggatgc?agaaggagat?cactgccctg 960

gcacccagca?caatgaagat?caagatcatt?gctcctcctg?agcgcaagta?ctccgtgtgg?1020

atcggcggct?ccatcctggc?ctcgctgtcc?accttccagc?agatgtggat?cagcaagcag?1080

gagtatgacg?agtccggccc?ctccatcgtc?caccgcaaat?gcttctag 1128

<210>6

<211>302

<212>PRT

<213 > people (Homo Sapiens)

<220>Homo?ALKBH4protein

<400>6

10 20 30 40 50 60

MAAAAAETPE?VLRECGCKGI?RTCLICERQR?GSDPPWELPP?AKTYRFIYCS?DTGWAVGTEE

70 80 90 100 110 120

SDFEGWAFPF?PGVMLIEDFV?TREEEAELVR?LMDRDPWKLS?QSGRRKQDYG?PKVNFRKQKL

130 140 150 160 170 180

KTEGFCGLPS?FSREVVRRMG?LYPGLEGFRP?VEQCNLDYCP?ERGSAIDPHL?DDAWLWGERL

190 200 210 220 230 240

VSLNLLSPTV?LSMCREAPGS?LLLCSAPSAA?PEALVDSVIA?PSRSVLCQEV?EVAIPLPARS

250 260 270 280 290 300

LLVLTGAARH?QWKHAIHRRH?IEARRVCVTF?RELSAEFGPG?GRQQELGQEL?LRIALSFQGR

302

PV

<210>7

<211>302

<212>PRT

<213 > people (Homo Sapiens)

<220>Homo?ALKBH4?H169A-D171A-H254A?mutation?protein

<400>7

10 20 30 40 50 60

MAAAAAETPE?VLRECGCKGI?RTCLICERQR?GSDPPWELPP?AKTYRFIYCS?DTGWAVGTEE

70 80 90 100 110 120

SDFEGWAFPF?PGVMLIEDFV?TREEEAELVR?LMDRDPWKLS?QSGRRKQDYG?PKVNFRKQKL

130 140 150 160 170 180

KTEGFCGLPS?FSREVVRRMG?LYPGLEGFRP?VEQCNLDYCP?ERGSAIDP AL? ADAWLWGERL

190 200 210 220 230 240

VSLNLLSPTV?LSMCREAPGS?LLLCSAPSAA?PEALVDSVIA?PSRSVLCQEV?EVAIPLPARS

250 260 270 280 290 300

LLVLTGAARH?QWK AAIHRRH?IEARRVCVTF?RELSAEFGPG?GRQQELGQEL?LRIALSFQGR

302

PV

<210>8

<211>300

<212>PRT

<213 > Mus (Mus Sapiens)

<220>Mouse?mALKBH4?protein

<400>8

10 20 30 40 50 60

MAAAAEVSLL?QECGCKGIRT?CLICERQRHR?DPPWQICLQK?KCCFLYCPDT?GWAAGAEGSD

70 80 90 100 110 120

LEGWAFPFPG?VTLIQDFVTP?EEEAEMVRLM?DCDPWKLSQS?GRKKQDYGPK?VNFRKQKLKM

130 140 150 160 170 180

AGFQGLPGFS?QKVVQRMGLY?PGLEDFQPVE?QCNLDYSPER?GSAIDPHLDD?AWLWGERLVS

190 200 210 220 230 240

LNLLSATVVS?MSPEAPGSLL?LCSAPSVRPD?AFEDSLVAPS?RSVPCQEVEV?AITVPRRSLL

250 260 270 280 290 300

VLTGAARHQW?THAIHRRHIK?ARRVCATFRE?LSSEFLPGGK?QQELGQELLQ?AALSFQGRPV

<210>9

<211>375

<212>PRT

<213 > people (Homo Sapiens)

<220>Homo?β-actin?protein

<400>9

10 20 30 40 50 60

MDDDIAALVV?DNGSGMCKAG?FAGDDAPRAV?FPSIVGRPRH?QGVMVGMGQK?DSYVGDEAQS

70 80 90 100 110 120

KRGILTLKYP?IEHGIVTNWD?DME mKIWHHTF?YNELRVAPEE?HPVLLTEAPL?NPKANREKMT

130 140 150 160 170 180

QIMFETFNTP?AMYVAIQAVL?SLYASGRTTG?IVMDSGDGVT?HTVPIYEGYA?LPHAILRLDL

190 200 210 220 230 240

AGRDLTDYLM?KILTERGYSF?TTTAEREIVR?DIKEKLCYVA?LDFEQEMATA?ASSSSLEKSY

250 260 270 280 290 300

ELPDGQVITI?GNERFRCPEA?LFQPSFLGME?SCGIHETTFN?SIMKCDVDIR?KDLYANTVLS

310 320 330 340 350 360

GGTTMYPGIA?DRMQKEITAL?APSTMKIKII?APPERKYSVW?IGGSILASLS?TFQQMWISKQ

370 375

EYDESGPSIV?HRKCF

<210>10

<211>375

<212>PRT

<213 > people (Homo Sapiens)

<220>Homo?β-actin?protein

<400>10

10 20 30 40 50 60

MDDDIAALVV?DNGSGMCKAG?FAGDDAPRAV?FPSIVGRPRH?QGVMVGMGQK?DSYVGDEAQS

70 80 90 100 110 120

KRGILTLKYP?IEHGIVTNWD?DME AIWHHTF?YNELRVAPEE?HPVLLTEAPL?NPKANREKMT

130 140 150 160 170 180

QIMFETFNTP?AMYVAIQAVL?SLYASGRTTG?IVMDSGDGVT?HTVPIYEGYA?LPHAILRLDL

190 200 210 220 230 240

AGRDLTDYLM?KILTERGYSF?TTTAEREIVR?DIKEKLCYVA?LDFEQEMATA?ASSSSLEKSY

250 260 270 280 290 300

ELPDGQVITI?GNERFRCPEA?LFQPSFLGME?SCGIHETTFN?SIMKCDVDIR?KDLYANTVLS

310 320 330 340 350 360

GGTTMYPGIA?DRMQKEITAL?APSTMKIKII?APPERKYSVW?IGGSILASLS?TFQQMWISKQ

370 375

EYDESGPSIV?HRKCF

<210>11

<211>26

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>

atagaattcccatggcggcggctgcc

<210>12

<211>27

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>12

accgtcgactcacacgggtcttccctg

<210>13

<211>25

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>13

ttcgaattctatggcggcggctgcc

<210>14

<211>27

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>14

gacctcgagatggcggcggctgccgcc

<210>15

<211>29

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>15

tataatgcggccgccacgggtcttccctg

<210>16

<211>29

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>16

cagctcgagatggatgatgatatcgccgc

<210>17

<211>35

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>17

ataagaatgcggccgcctagaagcatttgcggtgg

<210>18

<211>33

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>18

gccattgaccccgccctggccgacgcctggctg

<210>19

<211>31

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>19

ggcaccagtggaaggctgccatccaccgcag

<210>20

<211>34

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>20

ctgggacgacatggaggcaatctggcaccacacc

<210>21

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>21

attgggatccaagtgctctg

<210>22

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>22

gggtgtggctacaggaggta

<210>23

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>23

ctttggtcacagtgggtgtg

<210>24

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>24

tcagcggcagaggtagagat

<210>25

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>25

tgccgggattaaagatatgc

<210>26

<211>20

<212>DNA

<213 > artificial sequence

<220 > primer sequence

<400>26

gcaacccctctggtgtttta

<210>27

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for ALKBH4

<400>27

gaucccgggauugaaauga

<210>28

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for ALKBH4

<400>28

gggauugaaaugaggagca

<210>29

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for ALKBH4

<400>29

gggaucauuugagcuuaaa

<210>30

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for NM II

<400>30

gggccaacauugaaacaua

<210>31

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for NM II

<400>31

gugcuacaguuuggaaata

<210>32

<211>19

<212>RNA

<213 > artificial sequence

<220 > siRNA sequence is for NM II

<400>32

cucgggaugaggugauuaa

Claims (4)

1. Use of human actin K84me1 modified by monomethylation of the 84th lysine in the preparation of a drug for preventing or treating cancer, the human muscle actin K84me1 modified by monomethylation of the 84th lysine The amino acid sequence of actin K84me1 is shown in SEQ ID NO:9. 2. A cDNA sequence of the human actin K84me1 mutant modified by monomethylation of the 84th lysine, its nucleotide sequence is as one of the following nucleotide sequences: i) the nucleotide sequence shown in SEQ ID NO: 5; ii) a nucleotide sequence encoding a protein of the same amino acid sequence as the nucleotide sequence of i), but differing in nucleotide sequence due to the degeneracy of the genetic code; or iii) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:10. 3. A human actin K84me1 mutant protein modified by monomethylation of the 84th lysine, the amino acid sequence of which is shown in SEQ ID NO: 10. 4. Use of a human actin K84me1 antigen epitope modified by monomethylation of the 84th lysine in the preparation of a medicament for preventing or treating cancer, the monomethylation modification of the 84th lysine The amino acid sequence of the human actin K84me1 epitope is TNWDDMEmKIWHHTFY.

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