CN109055561A - LncRNA-AP003774.1 is diagnosing and/or treating the application in breast cancers - Google Patents
LncRNA-AP003774.1 is diagnosing and/or treating the application in breast cancers Download PDFInfo
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Abstract
本发明属于生物技术领域，尤其涉及lncRNA‑AP003774.1在诊断和/或治疗乳腺癌症中的应用。所述lncRNA‑AP003774.1在乳腺癌细胞中有非常明显的上调，可辅助用于乳腺癌的筛查。此外，本发明发现敲除lncRNA‑AP003774.1可有效抑制乳腺癌细胞的侵袭、转移和增殖，可用于治疗乳腺癌。
The invention belongs to the field of biotechnology, and in particular relates to the application of lncRNA-AP003774.1 in the diagnosis and/or treatment of breast cancer. The lncRNA-AP003774.1 is significantly up-regulated in breast cancer cells, and can be used as an aid in the screening of breast cancer. In addition, the present invention finds that knocking out lncRNA‑AP003774.1 can effectively inhibit the invasion, metastasis and proliferation of breast cancer cells, and can be used to treat breast cancer.
Description
Technical field
The invention belongs to field of biotechnology more particularly to lncRNA-AP003774.1 to diagnose and/or treat breast cancer
Application in disease.
Background technique
Breast cancer is most commonly seen one of the malignant tumour of global women, it is also the highest cancer of the death rate in female patient
Disease type.The new diagnosed case number of the annual women with breast cancer in China accounts for about global 12.2%, dead female due to breast cancer
Property patient accounts for about global 9.6%.With the progress of oncological clinical research and basic research, the various therapies of breast cancer without
Still all improve to some extent in therapeutic effect by safety, but in recent years the morbidity and mortality of global breast cancer still by
Step rises, so the Molecular Biology Mechanism of research breast cancer occurrence and development, realizes the diagnosis as early as possible to breast cancer and effectively control
It treats, is current clinical medicine worker and basic medical research personnel problem in the urgent need to address.
It is more than 200nt that long-chain non-coding RNA (long noncoding RNA, lncRNA), which is a kind of transcript length,
RNA molecule, themselves not coding protein, but in epigenetic regulation, transcriptional control and transcription in the form of RNA
The expression of the controlling gene in a variety of levels such as regulation afterwards.LncRNAs is initially considered as the by-product of rna plymerase ii transcription
Object has no biological function.However, recent studies indicate that, lncRNAs participates in chromatin modification, transcriptional activation, the interior fortune of core
A variety of important regulation processes such as defeated.More and more researches show that lncRNAs played in tumor development it is very important
Effect, this without suspected of disclose tumor development mechanism bring new hope.At present for lncRNA in breast cancer disease
Research in sick field is still at an early stage, and therefore, screening can have effective for the lncRNA for preventing and treating tumour
Important meaning.
Summary of the invention
In order to make up for the deficiencies of the prior art, an object of the present invention is to provide a kind of product of Diagnosis of Breast cancer, makes
Patient with breast cancer obtains medical treatment in early days, and then improves survival rate and quality of life.
The second object of the present invention is to provide a kind for the treatment of means and pharmaceutical composition, realize that the accurate molecule of breast cancer is controlled
It treats.
The third object of the present invention is to provide a kind of method of the potential drug of screening treatment breast cancer, and the drug can have
Effect reduces the expression of AP003774.1.
The purpose of the invention is achieved by the following technical solution:
The present invention provides the reagents of detection lncRNA-AP003774.1 expression to produce in preparation diagnosis early-stage breast cancer
Purposes in product.AP003774.1 gene is located at No. 11 chromosomes, and RNA length is that 2,340bps, sequence name is
ENST00000316124。
Further, the lncRNA-AP003774.1 expresses up-regulation in patient with breast cancer.
Further, the reagent is selected from the probe of specific recognition lncRNA-AP003774.1；Or specific amplification
The primer of lncRNA-AP003774.1.
The present invention also provides a kind of product for early diagnosing breast cancer, the product includes lncRNA- in detection sample
The reagent of AP003774.1 expression.As long as product of the present invention is able to detect that the expression of lncRNA-AP003774.1
, and be not limited to common testing product such as chip, calculate film item, preparation or kit etc.." sample " includes thin
Born of the same parents, tissue, organ, body fluid (blood, lymph etc.), digestive juice, urine, excrement etc..It is preferred that sample is tissue, blood.In this hair
In bright specific embodiment, selected sample is cell.
Further, the reagent includes the probe or specific amplification of specific recognition lncRNA-AP003774.1
The primer of lncRNA-AP003774.1.
In a specific embodiment of the invention, the reagent includes drawing for specific amplification lncRNA-AP003774.1
Object, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
The present invention also provides the purposes of lncRNA-AP003774.1, it is used to prepare the drug for preventing and treating breast cancer
Composition.
Described pharmaceutical composition includes the inhibitor of lncRNA-AP003774.1 functional expression, and the inhibitor can
Inhibit lncRNA-AP003774.1 or is related to the expression of the substance of the upstream lncRNA-AP003774.1 or downstream pathway.
Further, the inhibitor is able to suppress the RAN disturbing molecule of lncRNA-AP003774.1 expression, comprising:
ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisense nucleic acid, or can express or be formed and is described
ShRNA, siRNA, dsRNA, Microrna, antisense nucleic acid construction.
In a specific embodiment of the invention, selected inhibitor is shRNA or can express the carrier of shRNA, described
ShRNA sequence is as shown in NO.3~5 SEQ ID.
The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition includes lncRNA-AP003774.1 function
Property expression inhibitor, the inhibitor can DNA or RNA (i.e. gene product) level on work.
Further, described pharmaceutical composition may also include with its other medicine class of the inhibitor compatibility and can pharmaceutically connect
The carrier and/or auxiliary material received.
Further, the carrier includes but is not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiology
Salt, excipient, filler, coagulating agent and blender, interfacial agent, diffusant, defoaming agent.
The present invention also provides the purposes of lncRNA-AP003774.1 a kind of, prevent and treat breast cancer for screening
Potential substance.
Further, the step of potential substance of screening prevention or treatment breast cancer includes:
Using the potential substance processing expression or the system containing lncRNA-AP003774.1；
Detect the expression of lncRNA-AP003774.1 in the system；
Wherein, if candidate substances can reduce lncRNA-AP003774.1 expression or activity (preferably significantly reduce, such as it is low
20% or more, preferably low 50% or more, more preferably low 80% or more) then shows that the candidate substances are prevention or treatment mammary gland
The potential substance of cancer.
Further, system recited above includes but is not limited to: cell system, subcellular system, solution system, tissue
System, organ systems or animal system.
Beneficial effects of the present invention:
Present invention firstly discovers that a kind of lncRNA, i.e. lncRNA-AP003774.1 relevant to breast cancer development generation,
Find that its expression quantity in breast cancer cell is obvious by the expression of the fluorescence quantitative PCR detection lncRNA-AP003774.1
Higher than galactophore epithelial cell, and the expression quantity in the breast cancer cell of high invasive ability is apparently higher than the mammary gland of low invasive ability
Cancer cell；The migration and competence for added value of breast cancer cell can be inhibited by knocking out lncRNA-AP003774.1.The present invention is to breast cancer
Research have great theoretical value, for treatment breast cancer disease have very high application value.
Detailed description of the invention
Fig. 1 is fluorescence quantitative PCR detection lncRNA-AP003774.1 in people's normal mammary epithelial MCF-10A and cream
Expression in adenocarcinoma cell MCF-7, T47D, MDA-MB-231 and MDA-MB-453.
Fig. 2 is GV248 slow virus infected cell efficiency chart.
Fig. 3 is that HCS scratch experiment is detected with different time after the slow virus knockout intracellular AP003774.1 of MDA-MB-231
The variation of cell migration ability, wherein shCtrl indicates the MDA-MB-231, shlncRNA- after the unloaded virus of infection
AP003774.1 indicates the MDA-MB-231 after slow virus of the infection containing lncRNA-AP003774.1.
Fig. 4 shows the quantitative analysis to cell migration rate, and left figure indicates the change of mobility after two groups of cell scratches, right figure
Indicate the change that multiple is migrated after two groups of cell scratches.* P < 0.05.
Fig. 5 is that HCS scratch experiment is detected with group of cells after the slow virus knockout intracellular AP003774.1 of MDA-MB-231
The change of mobility and migration multiple after scratch, wherein shCtrl indicates the MDA-MB-231 after the unloaded virus of infection,
ShlncRNA-AP003774.1 indicates the MDA-MB-231 after slow virus of the infection containing lncRNA-AP003774.1, left figure table
Show the change of mobility after two groups of cell scratches, right figure indicates the change that multiple is migrated after two groups of cell scratches, * P < 0.05.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
AP003774.1 gene:
AP003774.1 is located at No. 11 chromosomes, and RNA length is that 2,340bps, sequence name is
ENST00000316124, AP003774 have 4 version sequences, and AP003774.1 is first version sequence of AP003774.This
Field is it will be recognized that practicability of the invention is not limited to any specific variants to target gene of the invention
Gene expression quantified.
The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not importances of the invention.
Chip, nucleic acid film item, kit:
In the present invention, chip includes: solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited
Needle, the oligonucleotide probe some or all of specifically correspond to shown in lncRNA-AP003774.1 sequence.It is described
Solid phase carrier includes inorganic carrier and organic carrier, and the inorganic carrier includes but is not limited to have silicon carrier, glass carrier, ceramics
Carrier etc.；The organic carrier includes polypropylene film, nylon membrane etc..
" probe " refers to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.Unless otherwise finger
Out, term " probe " is often referred to match by complementary base and combine with another polynucleotides (often referred to as " target polynucleotide ")
Polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence complementarity target it is more
Nucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system includes, but are not limited to: molten
Liquid phase, solid phase, mixed phase or in situ hybridization measuring method.
These probes have the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence
80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA,
Be also possible to RNA, furthermore it is possible to for part of it or whole nucleotide by PNA (Polyamide nucleicacid,
Peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked nucleic acid), ENA
(registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerolnucleic acid,
Glycerol nucleic acid), the obtained polynucleotides of the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid).
In the present invention, nucleic acid film item includes substrate and the oligonucleotide probe that is fixed in the substrate；The substrate
Can be any substrate suitable for immobilized oligonucleotide probe, for example, nylon membrane, nitrocellulose filter, polypropylene screen, sheet glass,
Silica gel chip, miniature magnetic bead etc..
The present invention provides a kind of kit, the kit can be used for detecting the expression water of lncRNA-AP003774.1
It is flat.The reagent of the expression for detecting lncRNA-AP003774.1 includes specificity in a specific embodiment of the present invention
The cDNA primer of lncRNA-AP003774.1 is expanded, the primer sequence is as shown in NO.1~2 SEQ ID.In addition, described
It may also include in kit for various reagents needed for extracting RNA, PCR, quantitative fluorescent PCR etc..
SEQ ID NO.1:5 '-TCTCCATCCTCACCACTGCTCAG-3 '；
SEQ ID NO.2:5 '-CATCTCTGCACCAGGCACTGTG-3 '.
Inhibitor and pharmaceutical composition:
Discovery based on inventor, the present invention provides the functional expression inhibitor of lncRNA-AP003774.1 a kind of,
The property of the inhibitor has no importance for the present invention, as long as it inhibits the functional expression of lncRNA-AP003774.1
, these inhibitor as lower lncRNA-AP003774.1 be useful substance, can be used for preventing or treating mammary gland
Cancer.
As a kind of preferred embodiment of the invention, the inhibitor of the lncRNA-AP003774.1 is a kind of lncRNA-
The siRNA molecule of AP003774.1 specificity.As used herein, " siRNA " refers to that one kind " can clone
To expression vector and express the DNA molecular of short RNA interfering (the RNA double-strand of siRNA, 19-21 nucleotide).It is effective in screening
ShRNA sequence when, the present inventor by largely compare analysis, to find out optimal effective segment.The present inventor's design
A variety of shRNA sequences have been synthesized, and they are transfected into breast cancer cell line by transfection reagent respectively and is verified, have selected interference
The optimal shRNA of effect is further tested in cellular level, is as a result proved effectively inhibit in cell the shRNA
The expression of lncRNA-AP003774.1 gene and the proliferation of breast cancer cell and invasive ability.
The present invention also provides a kind of pharmaceutical compositions, it contains a effective amount of lncRNA-AP003774.1's
Inhibitor and pharmaceutically acceptable carrier.The composition can be used for inhibiting liver cancer.Any lncRNA- above-mentioned
The inhibitor of AP003774.1 is used equally for the preparation of pharmaceutical composition.
In the present invention, pharmaceutically acceptable carrier includes but is not limited to buffer, emulsifier, suspending agent, stabilization
Agent, preservative, physiological saline, excipient, filler, coagulating agent and blender, interfacial agent, diffusant, defoaming agent.Such as this
Used in text, " effective quantity " refers to can generate function or active and can be received by people and/or animal to people and/or animal
Amount." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.It should
Term refers to medicament carriers some in this way: themselves not being necessary active constituent, and does not have excessive toxicity after applying.It closes
Suitable carrier is well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain liquid in the composition,
Such as water, salt water, buffer.In addition, there is likely to be complementary substance in these carriers, such as filler, lubricant help stream
Agent, wetting agent or emulsifier, pH buffer substance etc..Cell (host is thin) transfection reagent can also be contained in the carrier.
The present invention can use a variety of methods well known in the art by the inhibitor or its encoding gene or its medicine
Compositions deliver medicine to mammal.Including but not limited to: subcutaneous injection, intramuscular injection, for percutaneous administration of, administer locally to, be implanted into,
Sustained release is given；Preferably, the administration mode is that non-bowel is given.
Preferably, the means that gene therapy can be used carry out.For example, can be directly by the inhibition of lncRNA-AP003774.1
Agent delivers medicine to subject by such as the methods of injection；Alternatively, lncRNA-AP003774.1 can will be carried by certain approach
The ceneme (such as expression vector or virus etc. or siRNA or shRNA) of inhibitor be delivered on target spot, and be allowed to table
Up to active lncRNA-AP003774.1 inhibitor, concrete condition need to be depending on the type of the inhibitor, these are these
Known to the technical staff of field.
Pharmaceutical composition of the invention can further include one or more anticancer agents.In specific embodiments,
Pharmaceutical composition includes the compound and at least one chemotherapeutics of at least one inhibition lncRNA-AP003774.1 expression.For
Chemotherapeutics of the invention, including but not limited to: micro-pipe activator, alkylating agent, nti-neoplastic antimetabolite, platinum-like compounds, DNA-
Alkylating agent, antitumor antibiotics agent, antimetabolite, microtubule stabilizing agent, tubulin destabilizing agent, hormone antagonist are opened up
Flutter isomerase inhibitors, kinases inhibitor, HMG-COA inhibitor, CDK inhibitor, cyclin inhibitors, Guang day
The virus of protease inhibitors, metal protease inhibitors, antisense nucleic acid, triple helix DNA, aptamer and molecular modification,
Bacterium and exotoxin reagent.
Pharmaceutical acceptable carrier may include but be not limited to: virus, micro-capsule, liposome, nano particle or polymer and its any group
It closes.Relevant delivering carrier may include but be not limited to: liposome, biocompatible polymer (including natural polymer and synthesis
Polymer), lipoprotein, polypeptide, polysaccharide, lipopolysaccharides, artificial viral envelope, inorganic (including metal) particle and bacterium or disease
Malicious (such as baculoviral, adenovirus and retrovirus), bacteriophage, sticking grain or plasmid vector.
Pharmaceutical composition of the invention can also can be with the drug combination of other treatment liver cancer, other therapeutic compound
Main active constituent is administered simultaneously, or even is administered simultaneously in same composition.
Pharmaceutical composition of the invention can also be with individual composition or the dosage shape different from main active constituent
Formula individually gives other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds,
And other dosage can be administered alone.It over the course for the treatment of, can be according to the severity of symptom, the frequency of recurrence and treatment side
The physiologic response of case adjusts the dosage of pharmaceutical composition of the present invention.
Statistical analysis:
In a specific embodiment of the present invention, experiment be all to be completed according to being at least repeated 3 times, result data be all with
The mode of mean+SD indicates, using SPSS18.0 statistical software come for statistical analysis, difference between the two
It is different to be examined using t, it is believed that there is statistical significance as P < 0.05.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The differential expression of 1 QPCR of embodiment verifying AP003774.1 gene
1, the total serum IgE in each cell is extracted with TRIzol
1.1 exhaust normal mammary epithelial MCF-10A or breast cancer cell MDA-MB-231, T47D, MCF- in 6 orifice plates
1ml TRIzol is added in the culture solution of 7 cells, every hole, so that it is covered cell, then blown and beaten 3 times with suction pipe or sample injector, cell is answered
Cracking completely, is then transferred in centrifuge tube.
1.2 are added the chloroform (0.2mL chloroform is added in 1mL TRIzol) of 0.2mL, vibration in the centrifuge tube equipped with lysate
It swings on device sufficiently oscillation mixing 20 seconds, is placed at room temperature for 5 minutes.4 DEG C of 12000g are centrifuged 10 minutes, then draw containing the upper of total serum IgE
For layer water phase into a new centrifuge tube, every milliliter of TRIzol can about draw 0.6mL upper strata aqueous phase.Organic phase and middle layer contain
DNA and protein, should be avoided and touch.
1.3 additions and the isometric isopropanol of upper strata aqueous phase, it is reverse to mix for several times, precipitation at room temperature 5 minutes.12000g 4℃
Centrifugation 10 minutes, in the visible RNA precipitate of tube bottom.Supernatant is abandoned, 1mL75% ethyl alcohol is added by every milliliter of TRIzol, is gently overturned mixed
It is even, to clean RNA precipitate.4 DEG C of 12000g are centrifuged 2 minutes, discard liquid, do not abandon RNA precipitate carefully.Room temperature inversion is dried
5-10 minutes.
1.4 dissolutions: appropriate DEPC processing water, which is added, dissolves RNA precipitate.Deposit in -80 DEG C.
2, the first chain cDNA is synthesized using M-MLV reverse transcriptase.Synthesis reagent and condition such as table 1:
Table 1 synthesizes the first chain cDNA reagent and condition
3, with the expression quantity of AP003774.1 in the SYBR Green PCR kit for fluorescence quantitative detection cell of Bio-Rad.
The amplimer sequence of 3.1 design AP003774.1, sequence is as shown in NO.2~3 SEQ ID.
3.2 prepare 25 μ l reaction systems: SYBR Green polymerase chain reaction system 12.5 μ l, forward and reverse primer (5 μ
M) each 1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water.Operations are carried out on ice.Using GAPDH as internal reference.Often
3 parallel pipes are arranged in a sample, and all amplified reactions are repeated three times the above reliability to guarantee result.
Amplification program are as follows: 95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 35 circulations.With SYBR Green work
For fluorescent marker, PCR reaction is carried out on Light Cycler fluorescence real-time quantitative PCR instrument, by melt curve analysis analysis and
Electrophoresis determines purpose band, with 2–△△CTThe relative expression quantity of method calculating AP003774.1.
As a result as shown in Figure 1, it is thin by fluorescence quantitative PCR detection normal mammary epithelial MCF-10A and breast cancer
Expression in born of the same parents MCF-7, T47D, MDA-MB-231 and MDA-MB-453, lncRNA-AP003774.1 is in breast cancer as the result is shown
Expression quantity in cell is apparently higher than MCF-10A cell, and in the MDA-MB-231 and MDA-MB-453 of high invasive ability
Expression quantity is apparently higher than MCF-7, T47D cell of low invasive ability.
The silencing of 2 AP003774.1 gene of embodiment
By shRNA slow-virus infection breast cancer cell MDA-MB-231, AP003774.1 in successful knockout cell, and
Stable cell strain is filtered out, the MDA-MB-231 cell of infection RNAi slow virus is known as SiAP003774.1/MDA-MB-231, feels
Dye control slow virus MDA-MB-231 cell is known as Scr/MDA-MB-231.
AP003774.1shRNA slow virus is the customization of Shanghai Ji Kai genome company, container name GV248.
Make silencing target spot totally three of AP003774.1 gene, sequence is respectively as follows: NO.3~5 SEQ ID.
SEQ ID NO.3:5 '-ATCCTCAAGCTTGGCCCAAAT-3 '
SEQ ID NO.4:5 '-AAGCAGCCACAGTGGATCATA-3 '
SEQ ID NO.5:5 '-CTGCAGCCAGCCAGTTTGTTA-3 '
Efficiency of infection is as shown in Figure 2, the results showed that transfection efficiency reaches 80% or more, it was demonstrated that this virus can succeed silencing
AP003774.1 in MDA-MB-231 cell.
3 HCS Cell migration assay of embodiment
The intracellular lncRNA-AP003774.1 of MDA-MB-231 is knocked out with slow virus using the detection of HCS cell scratch experiment
The change of cell migration ability afterwards.
HCS cell scratch detection experimental procedure:
1. will be in each experimental group cell of logarithmic growth phase after pancreatin digests, complete medium is resuspended into cell suspension,
It counts；
2. plating cells number is set as 50000cell/well, next day cell of being subject to reaches 90% or more convergence degree.37
DEG C, 5%CO2Incubator culture, every group of 3 multiple holes, cultivating system are 100 holes μ L/.
3. second day changes low concentration blood serum medium, the lower center portion position of 96 orifice plates is directed at using scratching instrument, it is light upwards
It pushes away to form scratch.
4. gently rinse 2-3 times using serum free medium, low concentration blood serum medium is added, under an optical microscope with
Machine chooses the width of three fixed position record scratches, and 0h takes pictures.
5. in 37 DEG C, 5%CO2It is cultivated in incubator, selects right times to sweep plate with Celigo according to healing.
6. analyzing migration area with Celigo.
This experiment is in triplicate.Wherein shCtrl indicates the MDA-MB-231, shlncRNA- after the unloaded virus of infection
AP003774.1 indicates the MDA-MB-231 after slow virus of the infection containing lncRNA-AP003774.1.
As a result as shown in Figure 3: with the extension of incubation time, the migration of the cell of silencing lncRNA-AP003774.1 away from
It is obviously shortened from the migration distance than cellular control unit, the results showed that knocking out lncRNA-AP003774.1 can significantly reduce MDA-
The transfer ability of MB-231 cell.
Fig. 4 shows the quantitative analysis to cell migration rate, and left figure indicates the change of mobility after two groups of cell scratches, right figure
Indicate the change that multiple is migrated after two groups of cell scratches.* P < 0.05.As a result it further proves to knock out lncRNA-AP003774.1
It can significantly reduce the transfer ability of MDA-MB-231 cell.
4 HCS cell proliferation experiment of embodiment
The proliferative capacity of infection front and back cell is detected using HCS cell proliferation experiment.AP003774.1 is knocked out as the result is shown
It can inhibit the proliferative capacity of MDA-MB-231 cell；
HCS cell proliferation experiment step:
1. complete medium is resuspended into cell suspension after each experimental group cell tryptase enzymic digestion in logarithmic growth phase,
It counts.
2. plating cells number is set as 2000cell/well.Every group of 3 multiple holes, cultivating system are 100 holes μ L/, bed board process
In need to ensure that cell number is added in every hole consistent.37 DEG C, 5%CO2Incubator culture.
3. daily Celigo detection read plate was primary, continuous detection read plate 5 days since after bed board second day.
4. by adjusting the input parameter of analysis setting, be accurately calculated in scanning orifice plate every time with green
The quantity of the cell of color fluorescence；Statistics drawing is carried out to data, draws 5 days cell Proliferation curves.
As a result such as Fig. 5 shows that shCtrl group cell Proliferation is normal, and the 5th day proliferation times compared to first day reach 4.02
Times；ShlncRNA-AP003774.1 group (knocking out lncRNA-AP003774.1 groups of cells) cell Proliferation significantly slows, and the 5th day
Only 1.33 times of proliferation times compared to first day, the Fold change=3.01 of shCtrl by comparison.The result shows that knocking out
AP003774.1 can inhibit the proliferative capacity of MDA-MB-231 cell.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Weifang Medical College
<120>lncRNA-AP003774.1 is diagnosing and/or treating the application in breast cancers
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213>artificial sequence
<400> 1
tctccatcct caccactgct cag 23
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
catctctgca ccaggcactg tg 22
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
atcctcaagc ttggcccaaa t 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
aagcagccac agtggatcat a 21
<210> 5
<211> 21
<212> DNA
<213>artificial sequence
<400> 5
ctgcagccag ccagtttgtt a 21
Claims (10)
1. detecting purposes of the reagent of lncRNA-AP003774.1 expression in preparation diagnosis early-stage breast cancer product.
2. a kind of product for early diagnosing breast cancer, the product includes lncRNA-AP003774.1 expression water in detection sample
Flat reagent.
3. product according to claim 2, which is characterized in that the reagent includes specific recognition lncRNA-
The probe of AP003774.1 or the primer of specific amplification lncRNA-AP003774.1；
Preferably, the reagent includes the primer of specific amplification lncRNA-AP003774.1, the primer sequence such as SEQ ID
Shown in NO.1 and SEQ ID NO.2.
Purposes of the 4.lncRNA-AP003774.1 in the pharmaceutical composition that preparation prevents and treats breast cancer.
5. purposes according to claim 4, which is characterized in that described pharmaceutical composition includes lncRNA-AP003774.1
The inhibitor of functional expression, the inhibitor are able to suppress lncRNA-AP003774.1 or are related to lncRNA-AP003774.1
The expression of the substance of upstream or downstream pathway.
6. purposes according to claim 5, which is characterized in that the inhibitor is able to suppress lncRNA-AP003774.1
The RAN disturbing molecule of expression；
Preferably, the RAN disturbing molecule is shRNA, and sequence is as shown in NO.3~5 SEQ ID.
7. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition includes lncRNA-AP003774.1 functional expression
Inhibitor.
8. purposes according to claim 7, which is characterized in that described pharmaceutical composition may also include matches with the inhibitor
5 its other medicine class and pharmaceutically acceptable carrier and/or auxiliary material.
9.lncRNA-AP003774.1 the purposes in the potential substance that screening prevents and treats breast cancer.
10. purposes according to claim 9, which is characterized in that the step of the potential substance of screening prevention or treatment breast cancer
Suddenly include:
Using the potential substance processing expression or the system containing lncRNA-AP003774.1；With
Detect the expression of lncRNA-AP003774.1 in the system；
Wherein, if the candidate substances can reduce the expression or activity of lncRNA-AP003774.1, show that the candidate substances are
The potential substance of prevention or treatment breast cancer.
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| CN201811223427.2A CN109055561A (en) | 2018-10-19 | 2018-10-19 | LncRNA-AP003774.1 is diagnosing and/or treating the application in breast cancers |
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| CN201811223427.2A CN109055561A (en) | 2018-10-19 | 2018-10-19 | LncRNA-AP003774.1 is diagnosing and/or treating the application in breast cancers |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825595A (en) * | 2019-04-15 | 2019-05-31 | 德阳市人民医院 | LncRNA marker relevant to breast cancer and its detection primer and application |
| CN110592218A (en) * | 2019-10-22 | 2019-12-20 | 德阳市人民医院 | Biomarker for diagnosing and treating breast cancer |
| CN110684847A (en) * | 2019-04-15 | 2020-01-14 | 德阳市人民医院 | Application of biomarker related to breast cancer occurrence and development |
-
2018
- 2018-10-19 CN CN201811223427.2A patent/CN109055561A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825595A (en) * | 2019-04-15 | 2019-05-31 | 德阳市人民医院 | LncRNA marker relevant to breast cancer and its detection primer and application |
| CN110684847A (en) * | 2019-04-15 | 2020-01-14 | 德阳市人民医院 | Application of biomarker related to breast cancer occurrence and development |
| CN110592218A (en) * | 2019-10-22 | 2019-12-20 | 德阳市人民医院 | Biomarker for diagnosing and treating breast cancer |
| CN110592218B (en) * | 2019-10-22 | 2020-09-15 | 德阳市人民医院 | Biomarker for diagnosing and treating breast cancer |
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