CN109758576A - Use of DLC3 for preparing drugs for targeted therapy of gastric cancer - Google Patents

Abstract

The invention belongs to the field of functions and applications of genes and proteins, and relates to a new use of liver cancer deletion gene 3 (DLC3), in particular to the use of DLC3 for preparing a drug for targeting gastric cancer. The present invention provides the use of a drug for regulating DLC3 for preparing a drug for targeted treatment of gastric cancer. Further, the drug for regulating DLC3 is lovastatin or other statins. The present invention reveals the role of DLC3 in promoting gastric cancer metastasis from clinical phenomenon, cell function, and molecular mechanism, and discusses the mechanism of gastric cancer metastasis with DLC3 as the node, proving that the lipid-lowering drug lovastatin regulates the DLC3/MACC1 axis by regulating the DLC3/MACC1 axis. Inhibit gastric cancer progression. Therefore, DLC3 can be used as a new gastric cancer treatment target, and a drug for the targeted treatment of gastric cancer can be prepared. The existing lovastatin and other statins have the potential to be further developed as a targeted treatment for gastric cancer.

Description

DLC3 is used to prepare the purposes of the drug of targeted therapy gastric cancer

Technical field

The invention belongs to the functions and application field of gene and albumen, are related to the new application of liver cancer deficiency gene 3 (DLC3), The purposes of the drug of targeted therapy gastric cancer is used to prepare more particularly to DLC3.

Background technique

Liver cancer deficiency gene 3 (deleted in liver cancer-3, DLC3) DLC3 is a member of DLC family. DLC family is considered as a kind of tumor suppressor gene, but one of its member DLC3 rarely has research at present.Theoretically, DLC3 has and can make The structural domain of Rho GTPase inactivation, the activation energy of Rho GTPase trigger a series of downstream signals and promote in tumour cell generation Skin mesenchymal transformation (epithelial-mesenchymal transition, EMT).The past document also has been reported that DLC3 a variety of Tumour expression is lowered, and then activates RhoA.However, current clinical prognosis meaning or DLC3 of the either DLC3 in gastric cancer It is unclear with the relationship of metabolic stress or EMT.

EMT is considered as one of initial step of malignant cell infiltration and transfer of epithelium genesis and current stomach The research hotspot in cancer field.The stomach cancer cell that EMT occurs will be detached from being adhered to each other in close cancer nests tissue originally, shape At single, nonpolar, moveable fusiform cell.The formation of EMT needs Cell tracking to be broken, cytoskeleton weight Row, and with the degradation of extracellular matrix.Currently, the relationship of metabolic stress and EMT are not fully understood, it is necessary to further visit Regulated and control network of the rope between them.

Summary of the invention

The purpose of the present invention is to provide the new applications of liver cancer deficiency gene 3 (DLC3), more particularly to DLC3 for making The purposes of the drug of standby targeted therapy gastric cancer.

The present invention provides the purposes that the drug of regulation DLC3 is used to prepare the drug of targeted therapy gastric cancer.

The drug of the further feature of purposes according to the present invention, the regulation DLC3 is Lovastatin or other The drug of Statins.

The invention firstly discloses: DLC3 is lowered in gastric cancer, and the gastric cancer of DLC3 low expression is easier to recur, always when existence Between it is also shorter.Mechanism is further studied, the relationship of Metastasis of Gastric Cancer and energetic supersession is disclosed, and has found that following signal is logical Road: silencing DLC3 expression can promote (the Metastasis-associated in colon of colon cancer metastasis related gene -1 Cancer-1, MACC1) expression.It is thin to influence gastric cancer by regulation glucose metabolism and cell motility for DLC3/MACC1 axis The energy chemotactic of born of the same parents.The present invention arrives cell function from clinical picture, then arrives molecular mechanism, discloses DLC3 and promotes Metastasis of Gastric Cancer Effect, and inquired into using DLC3 as node the mechanism of Metastasis of Gastric Cancer, it was demonstrated that fat-reducing medicament Lovastatin passes through regulation DLC3/ MACC1 axis inhibits gastric cancer progress.Therefore, can be using DLC3 as new curing gastric cancer target spot, and prepare targeted therapy gastric cancer The drug of drug, existing Lovastatin and other Statins has the potential for the drug for being further developed as targeted therapy gastric cancer.

Detailed description of the invention

Fig. 1 shows that DLC3 inhibits proliferation of human gastric cancer cell and migration.

Fig. 2 is results of animal figure, and display DLC3 low expression promotes Metastasis of Gastric Cancer, influences glucose metabolism.

Fig. 3 shows the gastric cancer prognosis of DLC3/MACC1 axis joint effect.

Fig. 4 shows DLC3 negative regulation MACC1 expression.

Fig. 5 shows that DLC3/MACC1 axis by regulation stomach cancer cell metabolism, influences energy chemotactic.

Fig. 6 shows that Lovastatin inhibits gastric cancer progress by regulation DLC3/MACC1 axis.

Specific embodiment

One, experimental material

Tissue specimen: gastric cancer and cancer beside organism's qRT-PCR sample are obtained from 12 patients with gastric cancer by operation, when materials Between be 2 months 2015；For paraffin embedding sample by performing the operation what excision was obtained from 90 patients with gastric cancer, the materials time is 2015 Year.All patients being included according to 2010 standard of UICC/AJCC carry out clinicopathologic stage: 19 be the gastric cancer I phase, 37 For II phase of gastric cancer, 34 are the gastric cancer III phase.Sample is fixed with 10% formalin, paraffin embedding, slice.All patients pass through Radical surgery tumor resection lesion, and do not received any antineoplaston before materials.

Key instrument: SpectraMax5 multi-function microplate reader is purchased from U.S. Molecular Devices company, Odyssey Imaging system is purchased from LI-COR company, and LightCycler 480 is purchased from Roche, and inverted microscope is just being set microscope and is purchased from Olympus, FACSCanto II streaming systems are purchased from BD Biosciences.

Main agents and consumptive material: fetal calf serum, 1640 culture mediums are purchased from Hyclone company, and 2000 × PBS powder is purchased from Biosharp company.It EDTA, is that Solarbio company produces without EDTA pancreatin, Tris-Hcl (PH8.8 and PH6.8) is purchased from Shanghai Bai Sai Bioisystech Co., Ltd, TEMED, 30% polyacrylamide, ammonium persulfate, 10%SDS etc. prepare the reagent purchase of gel From Biosharp company.Tris alkali is purchased from Biosharp company.Glycine, SDS powder are the production of Sigma company.Methanol, isopropyl Alcohol, dehydrated alcohol, chloroform, tween are the production of Guangdong Province's chemical reagent engineering and technological research development centre.Skimmed milk power is adopted Purchased from Biosharp company.2000 × TBS powder, primary antibody dilution are purchased from Biosharp company.Holoprotein extracts kit is The production of Co., Ltd, Nanjing Keygen Biotech.5 × albumen sample-loading buffer is purchased from Hangzhou Fu De Biotechnology Co., Ltd.Rabbit Dan Ke Grand E-cadherin, vimentin and GAPDH are purchased from Cell Signal Technology, and the anti-DLC3 antibody of rabbit polyclonal is purchased from Proteintech company, the anti-MACC1 antibody of rabbit monoclonal are purchased from Abnova company, and the anti-Ki-67 antibody of rabbit monoclonal is purchased from Cell Signaling technology company, goat-anti rabbit secondary antibody are the production of Li-COR company.Dimethylbenzene, sodium citrate buffer, mistake Hydrogen peroxide solution, DAB working solution, haematoxylin, hydrochloric acid, neutral gum are purchased from the exploitation of Guangdong Province's chemical reagent engineering and technological research The heart.Each model Tissue Culture Dish, 6 orifice plates, 12 orifice plates, 96 orifice plates are Corning Incorporated's production.The cell Transwell is purchased from healthy and free from worry public affairs Department.BCA determination of protein concentration kit is the green skies biotech firm production in Jiangsu.Trizol Kit is purchased from Takara, and cDNA is closed Takara is purchased from Reverse Transcriptase kit.SYBR Green I Master kit is purchased from Takara.Apoptosis detection kit is Three arrow biological productions.ROS detection kit is the green skies biotech firm production in Jiangsu.EDU kit is sharp rich production.MTT purchase From Sigma company.Dimethyl sulfoxide is purchased from Guangzhou Hua Da company.Recombinant slow virus is purchased from Ji Kai company.

Two, experimental method

1. immunohistochemical staining

Roasting piece: slice is baked piece 3 hours on 65 DEG C of roasting piece machines

Dewaxing: twice through xylene soak, every time after ten minutes, successively through 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, which impregnate 3 minutes, to dewax, and is finally immersed in PBS solution and washs 3 times, every time 5 minutes.

Antigen retrieval: configuration citrate solution 2000ml is placed in pressure cooker, and slice is placed in slide holding frame and is put into In citrate solution, electromagnetic oven heating to pressure cooker jet starts timing, stops heating and being placed in pressure cooker cold after 3 minutes Recovery room temperature is cooled in water.Slice is taken out to wash 3 times, every time 3 minutes through PBS solution.

Endogenous peroxydase inactivation: slice impregnates 10 minutes in 3% hydrogenperoxide steam generator, continues to use PBS solution Washing 3 times, every time 3 minutes.

Closing: 5%BSA is added dropwise and is incubated at room temperature tissue 1 hour

It incubates primary antibody: primary antibody is added dropwise in tissue, is placed in wet box in 4 DEG C of refrigerators and is incubated overnight.

Rewarming: after wet box is transferred to normal temperature environment 1 hour by next day, with PBS solution washing slice 5 times, 3 minutes every time

It incubates secondary antibody: corresponding kind source property secondary antibody is added dropwise in tissue, is incubated at room temperature 1 hour, continues to be washed with PBS solution and cut Piece 5 times, every time 5 minutes.

DAB colour developing: press DAB concentrate: DBA substrate buffer solution=1:20 configures DAB solution, and drop is in tissue, and develop the color 3- Color development stopping in clear water is immersed after ten minutes.

Haematoxylin dyeing: the slice after colour developing is placed in haematoxylin solution after dyeing 10~15 minutes, under tap water Flowing water rinses about 10min.

Differentiation is anti-blue: it is to impregnate 2 seconds in l% hydrochloride alcohol that slice, which is placed in concentration, again, is transferred to tap water down-flow water rapidly It rinses about 5 minutes, in microscopically observation nuclear targeting situation, if unobvious repeatable haematoxylin dyeing and subsequent rinse, The step of differentiation.

It is transparent: histotomy of the dye after good being sequentially placed into 70%, 80%, 90%, 95% alcoholic solution and impregnates 2 points Clock, 100% alcohol impregnate about 10 minutes, are finally soaked in 10min in dimethylbenzene.

Mounting is taken pictures: the histotomy after transparent draws neutral tree fat drips with suction pipe after the natural air drying of ventilation and exists Wax disk(-sc) is placed in and just sets microscopically observation, takes pictures by tissue, covered, hardening of resin after placing 1-2 days at room temperature.

Evaluation criterion: dye level is divided into 0 point (colourless), 1 point (faint yellow), 2 points (yellow), 3 points (brown color), often Kind dyeing occupied area is then according to 0 point (0%), 1 point (1%-25%), 2 points (26%-50%), 3 points (51%-75%) or 4 points (76%-100%) scores.Dye level scoring is added after being multiplied with the scoring of corresponding stained area, has both been obtained last Dyeing scoring.Wherein it is defined as negative expression for 0-2 points, 3-5 points are defined as weakly positive expression, and 6-12 points are defined as strong positive table It reaches.

2.MTT experiment

Kind plate: need to be arranged blank group, negative control group, experimental group, 5 multiple holes of every group of setting by experiment.Cell is disappeared Change, precipitated after centrifugation, complete medium is added, cell is resuspended and counts, by the plantation of 2000/hole density in 96 orifice plates, Liquid volume is no more than 200ul in every hole, cultivates in 37 DEG C of incubators.

Processing: it handles after overnight incubation by experimental group to continue to be put in 37 DEG C of incubators after cell respectively and cultivate.

0.5% MTT stoste is made into MTT working solution by 1:9 with the RPMI-1640 culture medium without fetal calf serum.By 96 MTT working solution 200ul is added after cell conditioned medium sucks in orifice plate, is protected from light in 37 DEG C of incubators and is incubated for 3h, suck supernatant, every hole adds Enter 150ul dimethyl sulfoxide and measures each hole at 490nm wavelength using microplate reader after room temperature shakes 10min to precipitating dissolution OD value.

3. plate clone is tested

Kind plate: will be precipitated after cell dissociation, centrifugation, complete medium is added, cell is resuspended, train completely by every hole 2ml Feeding base, 1000 cells calculate the culture volume and cell number of needs, draw after mixing well corresponding celliferous complete Full culture medium kind is placed in 37 DEG C of incubator cultures in 6 orifice plates.

Processing: need to handle cell in each hole by experiment after adherent.

Fixed: culture sucked supernatant after 14 days, was washed 3 times with PBS, and the fixed cell 30min of 4% paraformaldehyde is added.

Dyeing: cells rinsed with PBS 3 times after fixed, 0.1% crystal violet solution 1ml is added and dyes 30min, clear water is washed Removal floating color is placed at dry and ventilated and dries, and camera is taken pictures.

4. active oxygen detects

Bed board: will be precipitated after cell dissociation, centrifugation, complete medium is added, cell is resuspended, complete by every hole 200ul Culture medium, 2000 cells calculate the culture volume and cell number of needs, draw after mixing well corresponding celliferous Complete medium kind is placed in 37 DEG C of incubator overnight incubations in 96 orifice plates.

Processing: next day needs to handle corresponding cell by experiment.

Load probe: according to DCFH-DA: serum-free medium=1:1000 dilutes DCFH-DA.Cell culture fluid is removed, The DCFH-DA that has diluted of 100ul is added in every hole, in being incubated for 20 minutes in 37 DEG C of cell incubators.Use serum-free cell culture Liquid washs cell three times, does not enter intracellular DCFH-DA with abundant removal.

Detection: it using fluorescence inverted microscope observation, takes pictures.

5. Apoptosis detects

Digestion, centrifugation: after needing to give cell respective handling by experiment, with without EDTA pancreatin by cell dissociation, from Supernatant is removed after the heart, is centrifuged again after PBS washing is added, is left and taken precipitating.

According to pure water |: 10 × Binding Buffer=9:1 configures Binding Buffer.Every solencyte is added 1ml and matches The Binding Buffer set is centrifuged after washing by 300g 10min, removes supernatant.

Cell concentration is adjusted to 1 × 10 with Binding Buffer⁶/ ml, and draw 100ul cell suspension and be placed in sample Guan Zhong.

Dyeing: 5ul Annexin V-FITC solution is added in every pipe, and after room temperature is protected from light incubation 10min, 5ul is added in every pipe PI solution, room temperature, which is protected from light, is incubated for 5min.

Detection: the every pipe of sample after dyeing is resuspended after 500ul PBS is added, in using flow cytomery sample in 1h.

6. Matrigel

Paving glue: Matrix stoste is diluted to 10%Matrix solution with the RPMI-1640 culture medium of serum-free.In upper chamber 100ul 10%Matrix solution is added, pipette tips are in advance in 4 degree of refrigerator cool overnights.2h is incubated for solution solidification in 37 DEG C of incubators For gel.

Bed board: will be precipitated after cell dissociation, centrifugation, and the RPMI-1640 culture medium without fetal calf serum is added and is resuspended Cell, by every hole 200ul complete medium, 1 × 10⁵A cell calculates the culture volume and cell number of needs, sufficiently mixed Corresponding cell suspension cover plant is drawn after even in the matrigel surface of upper chamber, the RPMI- for containing 10% fetal calf serum is added in lower room 1640 culture mediums, in 37 DEG C incubator culture 24-48 hours.

It is fixed: to take out cell after 48h and suck upper chamber liquid, the cell of film is secured across using 4% paraformaldehyde 30min。

Dyeing: 30min is dyed using 0.1% crystal violet solution.

As a result with inverted microscope observation, acquisition.

7. cell scratch experiment

Bed board: will be precipitated after cell dissociation, centrifugation, and the RPMI-1640 containing 10% fetal calf serum is added and cultivates base weight Cell is pressed 5 × 10 by outstanding cell⁵/ hole density kind enters in 6 orifice plates, is placed in 37 DEG C of incubator cultures.

Scratch: marking scratch with the vertical ware bottom of 200ul pipette tips after cell covers with ware bottom, washes away drift with PBS rinse ware bottom Floating cell is added the RPMI-1640 culture medium containing 2% fetal calf serum and is cultivated.

Observation: respectively at 0h, for 24 hours, 48h using inverted microscope observe scratch healing state, shooting, collecting.

8. quantitative fluorescent PCR

The extraction of 8.1RNA

Centrifuge is pre-chilled in half an hour in advance.The supernatant for discarding the cell of RNA to be mentioned is pre-chilled in right amount with suction pipe absorption PBS solution is added in adherent cell, jiggles and discards solution afterwards for several times, is repeated 3 times, for the last time need to be thorough with liquid-transfering gun Exhaust PBS.LmlTrizol solution is added, jiggling makes the entire ware bottom of Trizol solution uniform fold, and cell is placed in 4.C It is shaken slowly on shaking table.After about 20-30min, lysate is drawn with liquid-transfering gun and blows and beats cell aufwuchsplate repeatedly, liquid is transferred to 1.5ml is gone in the EP pipe of RNA enzyme.

The chloroform (chloroform) of 1/5 lysate volume (200u1) is added into EP pipe, gently overturns concussion up and down Lmin is stored at room temperature 5min.

EP pipe is placed in micro refrigerated centrifuge, 4 DEG C of centrifugation 15min of 12000rpm.

After the completion of centrifugation, the bright liquid (remaining to discard) in 350ul volume upper layer is carefully slowly drawn with liquid-transfering gun, The new standby 1.5ml capacity of injection is gone in enzyme EP pipe, then isometric aqueous isopropanol (350u1) is continuously added into the pipe, It is mixed by inversion about 1min repeatedly after covering EP pipe lid, is then allowed to stand about 10min.

EP pipe is placed in refrigerated centrifuge, 4 DEG C of centrifugation 10min of 12000rpm.

Supernatant liquid is discarded after centrifugation, 75% ethyl alcohol (dehydrated alcohol: DEPC water=3:1 is prepared) that 1ml pre-cooling is added is clear Wash precipitating.

In refrigerated centrifuge, 4 DEG C of centrifugation 5min of 12000rpm.

Supernatant liquid is carefully slowly drawn with liquid-transfering gun and is discarded, and avoids touching precipitating, then carefully drawn with filter paper It is retained in the micro liquid of tube wall, is finally placed in drying precipitating in Biohazard Safety Equipment.

Become transparent gel-shaped to visually precipitate, 5~15ul is added toward EP tube bottom and goes RNA enzyme water dissolution precipitating.

Use the purity and concentration of the proposed sample RNA of spectrophotometric determination.The zeroing of lul deionized water standard is inhaled first, Then the extracted RNA sample of lul, measurement result are inhaled.When entering 260/280 ratio numerical value greater than 1.8, then being included in research can be after The experiment of continuous next step, otherwise discards mentioned RNA sample, extracts again.Record the concentration of the RNA that all samples measure and pure Degree has marked title and the extraction time of sample, and 80 DEG C of refrigerators are given in placement can be reserved for half a year.

8.2 reverse transcription step

Reverse transcription using Japan Takara Reverse Transcriptase kit, by qualification RNA immediately reverse transcription at cDNA.It is reacted System is 10ul, as follows:

Remove RNA enzyme water	7ul
		5×PrimeScript RT Master Mix	2ul
RNA	1ul

Concentration is diluted to 500 units or so with deionized water by the RNA sample proposed, then each reverse transcription system The RNA sample 1ul is added, then calculates and needs to be added the volume of the deionized water of whole system polishing to 10ul.Reference table 2.4 sequentially add corresponding reagent in the EP pipe for going RNA enzyme with dosage in order, and slightly brief centrifugation is gathered in by all liq The tube bottom of EP pipe forbids bubble occur.It is immediately placed into reverse transcription machine, setting " 37 DEG C of 15min, 85 DEG C of 3s, 4 DEG C unlimited " Condition is reacted.After the completion of reverse transcription, 30ul is added into each small EP pipe respectively, the dilution mixing of enzyme water is gone to be placed on 4 DEG C Refrigerator cold-storage is spare.

The primer sequence is as follows in experiment:

8.3qRT-PCR

Reagent required for reaction system (10ul) is added according to following table carries out qRT-PCR:

Remove RNA enzyme water	3.6ul
		Upstream primer	0.2ul
Downstream primer	0.2ul
		Taq enzyme	5ul
cDNA	1ul

Prepare corresponding EP pipe respectively according to testing gene, is prepared respectively according to four kinds of reagents before upper table, after Taq enzyme is added All steps will be protected from light.By the transient liquid centrifugation in each EP pipe, it is uniformly mixed it then accurate with micropipette rifle 9ul is drawn on ground, and the hole wall along PCR amplification plate is squeezed into completely, is each all provided with 3 multiple holes, then dilute toward each hole corresponding 1ul of addition CDNA after releasing guarantees that additional amount is accurate.Finally in the dedicated pad pasting close adhesion plate of PCR plate, each loading hole is completely covered in guarantee, Then 1000rpm5min is centrifuged in centrifuge, finally PCR plate is put into Roche quantitative fluorescence PCR instrument, upper machine testing, is selected Specific amplification program is selected, computer is waited to export result.

9. immunofluorescence dyeing

Kind ware: supernatant will be removed after cell dissociation, centrifugation, is adjusted cell density to 1 × 10 with complete medium⁵A/ Ml, and 1ml cell suspension inoculation cell is drawn in the burnt dedicated ware of copolymerization, cell is carefully shaken up, 37 DEG C of incubator cultures are placed in.

After needing to handle cell by experiment, cell 2 times are cleaned with the PBS solution after filtration, each 5min.

The 4% fixed cell 30min of paraformaldehyde solution 400ul is added.

PBS is added to clean 3 times, it is every all over 5min.

0.5% triton x-100 of 400ul filtering PBS configuration, room temperature rupture of membranes 10min is added.

It continuously adds PBS to wash 3 times, each 5min.

5%BSA solution 400ul room temperature is added and closes 30min.

The primary antibody working solution that 5%BSA is prepared is added, is incubated overnight in 4 DEG C of refrigerators；E-cadherin,vimentin, MACC1 and DLC3 primary antibody dilution ratio is adjusted according to product description.

Next day takes out cell, sucks primary antibody working solution after being stored at room temperature rewarming 1h, and PBS is added and washs 5 times, each 3min.

The fluorescence secondary antibody working solution of corresponding kind source property is added, room temperature, which is protected from light, is incubated for 1h.

Secondary antibody working solution is discarded, PBS is added and washs 5 times, each 3min.

500ul 5ug/ml DAPI solution is added, is stored at room temperature dyeing 10min.

PBS is added to wash 5 times, each 3min.

The PBS solution of 400ul is eventually adding to keep ware bottom wet, is observed under Laser Scanning Confocal Microscope and acquires image.

10. zoopery

The BALB/c nude mice that zoopery is used is 4 week old, male, purchased from Nanfang Medical Univ's animal experiment study The heart.

10.1 mouse subcutaneous tumors models

Nude mice is grouped at random by experimental design, every group at least 5.Precipitating will be washed with PBS after cell dissociation, and with 1000rpm is centrifuged 3min, in triplicate.The cell for eliminating serum is prepared into 1 × 10⁸The cell suspension of a/ml, takes 100ul cell suspension is subcutaneously injected in mouse right hind root row.After being inoculated with subcutaneous tumors the 6th day, measurement mouse is swollen within every 2 days The longest diameter (L) and shortest diameter (W) of tumor, and calculate gross tumor volume V (cm³)=(L × W²The growth curve of subcutaneous tumors is made in)/2. After subcutaneous tumor formation 30 days, takes off cervical approach and put to death nude mice, collect subcutaneous tumors body tissue, weigh and photograph to record.

10.2 Lung metastases model

Nude mice is grouped at random by experimental design, every group at least 5.Cell precipitation will be resuspended with PBS after cell dissociation, and Supernatant is removed after being centrifuged 3min with 1000 turns/min, in triplicate.The cell for removing serum is prepared into 5 × 10⁷A/ml's is thin Born of the same parents' suspension.Mousetail is immersed in 1min in pre-prepd warm water, dress mouse enters fixator after tail veins expansion, makes tail It bar is exposed to outside fixator.Cotton ball soaked in alcohol disinfection tail portion is given, takes 100ul cell suspension to be slowly injected into mouse tail vein, after 4 weeks Mouse is put to death, collected, fixed mouse lung tissue and photograph to record.

10.3 liver metastasis models

Nude mice is grouped at random by experimental design, and every group at least 5.By after cell dissociation with PBS be resuspended cell precipitation, and with Supernatant is removed after 1000 turns/min centrifugation 3min, in triplicate.The cell for removing serum is prepared into 6 × 10⁷The cell of a/ml Suspension.Mouse takes right lateral position, makees an about 0.8cm skin incision in left arcus costarum lower edge, successively cuts off fascia and muscle, use is anodontia Tweezer gently clamps mouse spleen, pulls out and is exposed to outside body cavity, 100ul cell suspension is extracted with insulin needle, from spleen lower edge End parallel inserting needle about 0.3-0.5cm, slowly injects cell.It is given after having injected at alcohol cotton stick compressing spleen inserting needle one minute, Sutures are given again to sew up the incision.Postoperative second day rises and gives Lovastatin 100mg/kg weight stomach-filling, and mouse is put to death after 4 weeks, receives Collection, fixed mouse liver and spleen tissue simultaneously photograph to record.

One: DLC3 low expression of embodiment promotes proliferation of human gastric cancer cell and migration

For effect of the clear DLC3 in stomach cancer cell, inventor constructs DLC3 mistake using people's MKN45 stomach cancer cell The stomach cancer cell model of expression and silencing.Be overexpressed DLC3 obviously inhibit gastric cancer cell activity (Figure 1A), DNA synthesis (Figure 1B) and Clonality (Fig. 1 C).On the contrary, DLC3 expression is inhibited to be obviously promoted above-mentioned biological process.Show that DLC3 negativity regulates and controls stomach The proliferative capacity of cancer cell.

Surprisingly, inventor also found, DLC3 can also influence the form of stomach cancer cell.After being overexpressed DCL3, Cell shortens into coccoid, and cell major diameter and minor axis ratio are decreased obviously (Fig. 1 D).Conversely, cell becomes long after silencing DLC3 Shuttle shape fusiform, cell major diameter and minor axis ratio significantly increase (Fig. 1 D).This illustrates that DLC3 participates in cytoskeleton and polar tune Section also prompts DLC3 to be likely to participate in the adjusting of cell movement.

In terms of cell movement function, inventor carried out scratch experiment (Fig. 1 E) and Transwell experiment (Fig. 1 F) into Row verifying.Being overexpressed DLC3 obviously inhibits stomach cancer cell to migrate and invade, and silencing DLC3 promotes stomach cancer cell migration and invasion. Then, in terms of molecular phenotype, same verifying is had also been made in inventor, is overexpressed DLC3 and stomach cancer cell is promoted to turn to epithelial phenotype Change, and silencing DLC3 promotes stomach cancer cell to mesenchyma Phenotypic Change (Fig. 1 G).

Embodiment two: zoopery proves that DLC3 low expression promotes Metastasis of Gastric Cancer, influences glucose metabolism

On the basis of cell model, the verifying of inventor's further progress zoopery.Transplanted Gastric Carcinoma is seeded in Nude mice by subcutaneous (Fig. 2A), silencing DLC3 obviously inhibit the growth rate (Fig. 2 B) of Transplanted Gastric Carcinoma.Taken at the 34th day transplantable tumor into Row measurement, either size and weight (Fig. 2 C), or tissue Ki-67 expression (Fig. 2 D), silencing DLC3 are obvious more right It is vigorous according to group growth.

In terms of transfer ability, inventor has carried out tail vein injection pulmonary metastases model and spleen injection liver metastasis model It is verified.By nude mice tail vein injection tumour cell, find the transfer stove number of MACC1 silencing group obviously compared with control group height (E and G of Fig. 2), and the incidence (Fig. 2 F) of Lung metastases is also apparently higher than control group.Liver metastasis model is injected by spleen, It was found that growth rate of the MACC1 silencing group in spleen original position is apparently higher than control group (Fig. 2 H), and liver and peritonaeum occur to turn The probability of shifting also greatly increases (Fig. 2 I and K).It is furthermore interesting that being imaged by small animal position emission tomography (PET)/CT, discovery DLC3 silencing group turns The glucose metabolism for moving stove is (Fig. 2 H) being remarkably reinforced, and prompts DLC3 it is also possible to influencing stomach cancer cell glucose metabolism.

Three: DLC3/MACC1 axis joint effect gastric cancer prognosis of embodiment

In order to understand the relationship of DLC3 and MACC1, inventor carries out bioinformatic analysis.TCGA gastric cancer data analysis knot DLC3 and MACC1 is in obvious negative correlativing relation (Fig. 3 A) to fruit as the result is shown.Inventor further collects the I-III phase in Nanfang Hospital The postoperative clinical patients sample of Radical Operation of Gastric Carcinoma is detected, and immunohistochemical analysis result also prompts MACC1 and DLC3 in albumen table Up to above showing negative correlativing relation (Fig. 3 B), and as staging is incremented by, the Proportion of patients of DLC3 feminine gender is also gradually risen (Fig. 3 C).In the patient of DLC3 feminine gender, the ratio of MACC1 positive expression is higher (Fig. 3 D), and DLC3 feminine gender and MACC1 sun The risk recurred in the patient of property is higher (E and F of Fig. 3).In terms of follow-up of surviving, the stomach of DLC3 feminine gender, MACC1 positive expression Cancer patient's recurrence-free survival phase is short, and DLC3 is positive, patient's recurrence-free survival phase of MACC1 feminine gender is longer.This shows DLC3/ MACC1 axis joint effect gastric cancer prognosis.

Example IV: DLC3 passes through RhoA/JNK/AP-1 transcriptional control MACC1

For the relationship of clear MACC1 and DLC3 upstream and downstream, inventor carries out the mRNA of discovery MACC1 after silencing to DLC3 Expression (Fig. 4 A) and protein expression (Fig. 4 B) obviously increase.Speculate that RhoA/JNK/AP-1 may be potential tune by the past document Control the access of MACC1 expression.For the molecule on access, it is separately added into the inhibitor of RhoA, ROCK, JNK, finds MACC1's MRNA expression is obvious to lower (Fig. 4 C).In protein level, after above-mentioned inhibitor is added on the basis of silencing DLC3, silencing DLC3 Caused MACC1 up-regulation is effectively blocked (Fig. 4 D), shows that DLC3 regulates and controls MACC1 transcription by RhoA/JNK/AP-1 negativity. In terms of cell function, Clone formation caused by silencing DLC3 increase (Fig. 4 E) and invasive ability enhancing (Fig. 4 F) can effectively Jnk inhibitor is blocked.Show that DLC3 influences downstream biological effect by RhoA/JNK/AP-1.

Five: DLC3/MACC1 axis of embodiment influences energy chemotactic by regulation stomach cancer cell metabolism

It is tested and is found by EdU, after being overexpressed MACC1, inhibit quilt as being overexpressed the increment of gastric cancer caused by DLC3 originally (Fig. 5 A) is reversed in part.The ability decline of glucose uptake after overexpression DLC3, after being overexpressed MACC1 on this basis, Portugal Restore (Fig. 5 B) in grape Sugar intake part.In addition, can be seen that DLC3 from the culture medium color and pH value of cell conditioned medium inhibits acid Substance generates, and MACC1 overexpression can make acidic materials increase (Fig. 5 C).This prompt DLC3/MACC1 axis influences the Portugal of stomach cancer cell Grape glycolysis.

It is influenced by cell metabolism, generates two aspect of apoptosis caused by with metabolic stress in active oxygen in stomach cancer cell, DLC3/MACC1 axis all shows regulating and controlling effect (Fig. 5 D-E).In addition, inventor also found that DLC3/MACC1 axis can influence gastric cancer The migration and invasion (Fig. 5 F-G) of cell.

To sum up, it is contemplated that DLC3/MACC1 axis influences gastric cancer metabolism and cell movement simultaneously, can speculate DLC3/MACC1 axis It may also influence the energy chemotactic effect of stomach cancer cell.It is tested by Transwell, upper chamber is answered with glucose deprivation simulation metabolism Swash, lower room is with the well-off state of high sugared analog energy.As a result meet expection, DLC3/MACC1 axis passes through regulation metabolism, shadow Stomach cancer cell is rung from sugar-free to the energy chemotactic effect of high sugar.

Embodiment six: Lovastatin inhibits gastric cancer progress by regulation DLC3/MACC1 axis

The past document report Lovastatin can inhibit MACC1 to transcribe, and can the present embodiment research Lovastatin influence The DLC3 of the upstream MACC1 is expressed.

It is found through experiments that, after the Lovastatin stimulation of various concentration is added in stomach cancer cell, the mRNA of DLC3 is with concentration Gradient expression gradually increases, and the expression of MACC1 is gradually reduced (Fig. 6 A).Also demonstrate that Lovastatin can promote in protein level Into DLC3 and inhibit MACC1 (Fig. 6 B).Then, being inoculated with liver metastasis model by spleen is confirming that Lovastatin can in body level Inhibit plantation stove tumour growth (Fig. 6 C), and inhibits the generation (Fig. 6 D) of hepatic metastases.The DLC3's and MACC1 of tumor tissues is immune Histochemical staining result is also shown, and Lovastatin promotes DLC3 expression, inhibits MACC1 expression (Fig. 6 C-D).It is stimulated in Lovastatin Lower silencing DLC3, the invasive ability (Fig. 6 E) of repressed cell and clonality (Fig. 7 F) are partially restored originally； Apoptosis caused by metabolic stress obtains part and alleviates (Fig. 6 G).Therefore, as the upstream RhoA inhibition molecule, DLC3 may It is played an important role in tumor metabolic and transfer by influencing MACC1, Lovastatin may be the treatment of targeting up-regulation DLC3 Property drug.

Therefore can be using DLC3 as new curing gastric cancer target spot, and the drug of targeted therapy gastric cancer is prepared, existing Lip river is cut down The drug of statin and other Statins has the potential for the drug for being further developed as targeted therapy gastric cancer.

Claims (2)

1. Use of a drug for regulating DLC3 for preparing a drug for targeted therapy of gastric cancer. 2. purposes according to claim 1 is characterized in that: the medicine that regulates DLC3 is lovastatin or other statins medicines.