CN108085389B

CN108085389B - LncRNA related to breast cancer and application thereof

Info

Publication number: CN108085389B
Application number: CN201711475307.7A
Authority: CN
Inventors: 石小峰; 孙锦云
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-06-09
Anticipated expiration: 2037-12-29
Also published as: CN108085389A

Abstract

The invention discloses a lncRNA related to breast cancer and application thereof, wherein the gene is LOC100507053, the expression of LOC100507053 in a breast cancer patient is found to be remarkably reduced for the first time, ROC analysis finds that LOC100507053 has a higher AUC value, which indicates that LOC100507053 has higher accuracy when applied to diagnosis of the breast cancer, and in vitro cell experiments prove that LOC100507053 is related to invasion and migration of the breast cancer, which indicates that LOC100507053 can be applied to treatment of the breast cancer as a molecular target.

Description

LncRNA related to breast cancer and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to lncRNA related to breast cancer and application thereof, in particular to LOC 100507053.

Background

Breast cancer is the most common malignant tumor of women worldwide, accounts for about 22 percent of all malignant tumors of women, and is one of the most major diseases endangering the health of women. At present, about 15 percent of the newly increased female malignant tumor cases in China are breast cancer cases, and the breast cancer becomes the female malignant tumor with the highest morbidity in China and is also the leading cause of death of women under the age of 45 due to the malignant tumor. More seriously, research data show that the incidence and mortality of breast Cancer are on the rise in China, and the health and the life of the majority of women are seriously harmed (Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China,2015.CA Cancer J Clin 2016; 66: 115-32.). The morbidity and mortality of solid tumor patients is generally caused by the disruption of normal body functions by disseminated tumor cells, and tumor cell migration has become a major hotspot in the study of potential mechanisms of cancer metastasis (Palmer TD, Ashby WJ, Lewis JD, Zijlstra A. adv Drug Deliv Rev 2011; 63: 568-81). Compared to primary tumors, metastatic tumors cannot be surgically removed and are resistant to chemotherapy, with 90% of Cancer deaths being caused by distant metastasis of the tumor (Fidler IJ. nat Rev Cancer 2003; 3: 453-8.). However, the molecular mechanisms mediating breast cancer cell migration are not well defined, and molecular markers that can predict their progression metastasis remain limited. The discovery of key molecules and regulation pathways in the process of breast cancer progression and metastasis is the key point in breast cancer research, and the method is helpful for guiding early diagnosis and treatment of breast cancer so as to reduce the fatality rate and improve prognosis.

High throughput studies have revealed that only a small proportion of the mammalian genome is transcribed as protein-coding genes (integrated encyclopedia of DNA elements in the human genome. Nature 2012489:57-74.) and that the majority is transcribed as non-coding RNA. Long non-coding RNAs (1 ncRNA) are a class of RNA molecules which are not protein-coding and have a length of more than 200bp and are discovered in recent years (Prensner JR, ChinnaiyaM. the expression of 1ncRNAs in cancer biology. cancer Discov 2011; 1: 391. 407). Many documents report that lncRNA plays an important role in the process of tumor progression metastasis: the differential expression of lncRNA as reported in CN201710522240.1, CN201710522694.9, CN201710522693.4 is related to the metastasis of liver cancer.

Although the five-year survival rate of the breast cancer after operation is improved to about 98% by the strategies of early detection, early treatment, early operation and the like, the metastasis and recurrence of the breast cancer still cannot be treated. More and more lncRNA molecules are proved to be related to the occurrence and development of the breast cancer, and the intensive research of lncRNA provides a new idea for the diagnosis and treatment of the breast cancer.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide lncRNA related to the occurrence and development of breast cancer, so as to provide a molecular target for the diagnosis and treatment of the breast cancer and realize the personalized diagnosis and treatment of patients.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a reagent capable of detecting the expression level of the LOC100507053 gene.

Further, the agent is selected from:

a probe specifically recognizing LOC 100507053; or

Primers for specifically amplifying LOC 100507053.

Furthermore, the primer sequence of the specific amplification LOC100507053 gene is shown in SEQ ID No. 1-2.

According to a second aspect of the invention there is provided a kit comprising the reagents of the first aspect of the invention.

In a third aspect, the invention provides a chip comprising a reagent according to the first aspect of the invention.

In a fourth aspect of the invention, there is provided a pharmaceutical composition comprising an enhancer of LOC 100507053.

Further, the promoter is an expression vector containing LOC 100507053.

Furthermore, the pharmaceutical composition also comprises other medicines compatible with the accelerant and pharmaceutically acceptable carriers and/or auxiliary materials.

A fifth aspect of the present invention provides a method of screening for a potential substance for preventing or treating breast cancer, the method comprising:

treating a system expressing or containing the LOC100507053 gene with a substance to be screened; and

detecting the expression of the LOC100507053 gene in said system;

wherein, if the substance to be screened can promote the expression or activity of the LOC100507053 gene, the screening substance is a potential substance for preventing or treating the breast cancer.

A sixth aspect of the invention provides the use of any one of:

a. the use of a reagent according to the first aspect of the invention in the manufacture of a product for diagnosing breast cancer;

b. the kit of the second aspect of the invention is used for preparing a product for diagnosing breast cancer;

c. the chip of the third aspect of the invention is applied to the preparation of products for diagnosing breast cancer;

d. the application of the pharmaceutical composition of the fourth aspect of the invention in preparing products for treating breast cancer;

e. the application of the pharmaceutical composition of the fourth aspect of the invention in preparing a product for treating breast cancer invasion;

f. the application of the pharmaceutical composition of the fourth aspect of the invention in preparing products for treating breast cancer metastasis;

application of LOC100507053 in screening potential substances for treating breast cancer.

Drawings

FIG. 1 is a graph showing the detection of the expression of LOC100507053 gene in breast cancer tissues by QPCR;

FIG. 2 is a graph showing the detection of the expression of the LOC100507053 gene in breast cancer cell lines by QPCR;

FIG. 3 is a graph of the transfection of LOC100507053 in breast cancer cells using QPCR;

FIG. 4 is a diagram showing the effect of LOC100507053 gene on breast cancer cell proliferation measured by the CCK-8 method;

FIG. 5 is a graph of the effect of LOC100507053 on breast cancer cell migration and invasion, as detected using a Transwell cell; wherein panel a is a graph of the effect of LOC100507053 on breast cancer cell migration; panel B is a graph of the effect of LOC100507053 on breast cancer cell invasion.

Detailed Description

According to the invention, through extensive and intensive research, the expression of lncRNA in a breast cancer specimen in a tumor tissue and a tissue beside the tumor is detected by adopting an lncRNA chip which covers a database most widely at present through a high-throughput method, lncRNA with obvious expression difference is found, and the relation between the lncRNA and the occurrence and development of the breast cancer is discussed, so that a better way and a better method are found for the diagnosis and the targeted therapy of the breast cancer. Through screening, the invention discovers that LOC100507053 is remarkably reduced in breast cancer for the first time. Experiments prove that the proliferation and invasion of breast cancer cells can be effectively inhibited by improving the expression level of LOC 100507053.

Biomarkers

In the present invention, "biomarker", "gene marker" and "molecular marker" may be used interchangeably and are any gene whose expression level in a tissue or cell is altered compared to the expression level in a normal or healthy cell or tissue.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention.

LOC100507053 gene

LOC100507053 is located in zone 3 of human staining longarm 2, No.4, and the nucleotide sequence of a representative human LOC100507053 gene is shown in LOC100507053 gene (NR _037884.1) in GeneBank, the current International public nucleic acid database. The full-length sequence of LOC100507053 nucleotide or a fragment thereof of the present invention can be obtained by PCR amplification, recombinant method, or synthetic method. LOC100507053 in the present invention includes wild type, mutant type or a fragment thereof.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly known as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target nucleic acid sequence, transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of the target nucleic acid sequence under substantially constant conditions of temperature, ionic strength, and pH, where multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification, commonly known as NASBA, amplification of the probe molecule itself using RNA replicase (commonly known as Q β replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip and kit

The present invention provides products including (but not limited to) formulations, chips or kits for detecting the expression level of the LOC100507053 gene in a subject. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown by LOC 100507053.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PNA, LNA, ENA, GNA, TNA, etc.

The invention provides a kit which can be used for detecting the expression of LOC 100507053. Preferably, the preparation or the kit further comprises a marker for marking the RNA sample, and a substrate corresponding to the marker. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit also comprises an instruction manual and/or chip image analysis software.

Accelerator and pharmaceutical composition

Based on the findings of the present invention, the present invention provides a pharmaceutical composition comprising an enhancer of LOC 100507053.

The promoter of LOC100507053 refers to any substance that can improve the stability of LOC100507053 gene or expression product, up-regulate the expression of LOC100507053, increase the effective duration of lncRNA LOC100507053, or promote the transcription of LOC100507053 gene, and these substances can be used in the present invention, and as a substance useful for up-regulating the expression of LOC100507053 gene, it can be used for preventing or treating lung adenocarcinoma.

In a preferred embodiment of the present invention, the promoter of LOC100507053 is an expression vector containing LOC 100507053. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, there are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The expression vector can be introduced into the host cell by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

The term "host cell" includes prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, such as a CHO cell, a COS cell, or the like.

Pharmaceutical composition

The pharmaceutical composition comprises the accelerant of LOC100507053, and/or other medicines compatible with the accelerant, and a pharmaceutically acceptable carrier and/or auxiliary materials.

The pharmaceutically acceptable carrier may be one or more, and includes, but is not limited to, diluents such as lactose, sodium chloride, glucose, urea, starch, water, and the like; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell (host cell) transfection reagent.

The present invention may employ various methods well known in the art for administering the promoter or gene encoding the promoter, or pharmaceutical composition thereof, to a mammal. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In a specific embodiment, the pharmaceutical composition comprises at least one compound that inhibits the expression of the LOC100507053 gene and at least one chemotherapeutic agent. Chemotherapeutic agents useful in the methods of the invention include, but are not limited to, DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial and exotoxin agents.

The pharmaceutical compositions of the invention may also be used in combination with other agents for the treatment of breast cancer, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The pharmaceutical compositions of the present invention may also be administered separately with other therapeutic compounds, either as separate compositions or in different dosage forms than the primary active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

Drug screening

The invention provides a method for screening a medicine for preventing or treating breast cancer, which comprises the following steps:

in the experimental group, a test compound was added to the culture system, and the expression level of LOC100507053 was determined; in the control group, no test compound was added to the same culture system, and the expression level of LOC100507053 was determined; wherein, if the expression level of LOC100507053 in the experimental group is higher than that in the control group, the substance to be screened is a potential substance of LOC 100507053.

In the present invention, the method further comprises: the potential substance obtained in the above step is further tested for its effect of inhibiting breast cancer, and if the test compound has a significant effect of inhibiting breast cancer, the compound is a potential substance for preventing or treating breast cancer.

Such culture systems include, but are not limited to, cell systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems (e.g., animal models, preferably non-human mammalian animal models, such as mice, rabbits, sheep, monkeys, etc.), and the like.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of Gene markers associated with Breast cancer

1. Sample collection

The samples of the paracarcinoma tissues and the breast cancer tissues of 8 cases of breast cancer patients with definite pathological diagnosis are collected, the conditions of the sample names, the tissue types, the numbers, the sampling dates, the sample processing processes and the like are written, the patients sign an informed consent, and all the samples are obtained by the consent of the tissue ethics committee.

2. Preparation of RNA sample (manipulation Using tissue RNA extraction kit of QIAGEN)

RNA samples were extracted using QIAGEN's tissue RNA extraction kit, and the specific procedures are described in the specification.

3. Reverse transcription and labelling

mRNA was reverse-transcribed into cDNA using the Low RNA Input Linear Amplification Kit, and the experimental group and the control group were labeled with Cy3, respectively.

4. Hybridization of

The gene chip adopts Kangcheng organism-Human lncRNA Array, and hybridization is carried out according to the steps of the chip use instruction.

5. Data analysis

Chip results are analyzed by using Agilent GeneSpring software, and lncRNA with significant difference (standard is that the difference of the expression quantity of lncRNA in cancer and nearby cancer is more than 2 times, and p is less than 0.05) in expression quantity is screened.

6. Results

The results show that LOC100507053 exhibits differential expression in breast cancer patients, with a significant down-regulation of expression levels in cancer tissues compared to paracancerous tissues.

Example 2 QPCR sequencing verification of differential expression of the LOC100507053 Gene

1. Large sample QPCR validation was performed on LOC100507053 gene differential expression. 50 cases of the para-breast cancer tissues and breast cancer tissues were selected according to the sample collection method in example 1.

2. RNA extraction

3、QPCR

1) Reaction system:

1 mul of RNA template, 1 mul of random primer and 12 mul of double distilled water are added, mixed evenly, centrifuged at low speed, and cooled on ice at 65 ℃ for 5 min.

The following ingredients were added successively to 12. mu.l of the reaction:

5 × 4. mu.l of reaction buffer, 1. mu.l of RNase inhibitor (20U/. mu.l), 2. mu.l of 10mM dNTP mixture, 1. mu.l of AMV reverse transcriptase (200U/. mu.l); fully and uniformly mixing and carrying out centrifugal treatment;

2) conditions for reverse transcription

25℃5min，42℃60min，70℃5min。

3) Polymerase chain reaction

Designing a primer:

QPCR amplification primers were designed based on the coding sequences of the LOC100507053 gene and GAPDH gene in Genebank, and were synthesized by Bomader Biometrics. The specific primer sequences are as follows:

LOC100507053 gene:

the forward primer is 5'-TTAGAGAAGGACAAGAATAG-3' (SEQ ID NO. 1);

the reverse primer was 5'-CAATATGAACAGACAACAG-3' (SEQ ID NO. 2).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 3);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 4).

Preparing a PCR reaction system:

2 XqqPCR mixture 12.5. mu.l, gene primer 2.0. mu.l, reverse transcription product 2.5. mu.l, ddH₂O 8.0μl。

And (3) PCR reaction conditions: extension reaction at 95 deg.C for 10min, (95 deg.C for 15s, 60 deg.C for 60 s). times.40 cycles, and 60 deg.C for 5 min. The temperature is raised to 1 ℃ every 20s at 75 ℃ to 95 ℃, and a dissolution curve is drawn. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

4. ROC curve analysis

Analysis of the subject working characteristics of LOC100507053 using the pROC package in the R language, calculation of two accurate confidence spaces, and plotting of ROC curves.

5. Statistical method

The experiments were performed in 3 replicates, the data were presented as mean ± sd, statistically analyzed using SPSS18.0 statistical software, and the paired comparison of cancer to paracancerous tissue was performed using t-test, which was considered statistically significant when P < 0.05.

6. Results

The QPCR result is shown in figure 1, compared with the breast cancer paracancerous tissue, LOC100507053 has the expression down-regulated effect in the breast cancer tissue, the difference has the statistical significance (P <0.05), and the result is consistent with the chip detection result; the ROC curve result shows that the AUC value of LOC100507053 is as high as 0.955, which has higher specificity and sensitivity, and suggests that LOC100507053 has higher accuracy when applied to the diagnosis of breast cancer.

Example 3 expression of LOC100507053 in breast cancer cell lines

1. Cell culture

Culturing human breast cancer cell lines MCF-7, SK-BR-3, MDA-MB-231 and a normal mammary gland epithelial cell line MCF-L0A, wherein the MDA-MB-231 is cultured in an L15 culture medium containing 10% fetal bovine serum, the SKBR3 is cultured in an RPMI-1640 culture medium containing 10% fetal bovine serum, and the MCF-7 and the normal mammary gland epithelial cell line MCF-10A are cultured in a DMEM culture medium containing 10% fetal bovine serum. 1% P/S was added to the culture. At 37 deg.C, 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. Extraction and concentration determination of RNA

1) Washing the cells to be collected with PBS, adding a proper amount of RNAioso Plus, incubating on ice for 2min for lysis, scraping the cells at the bottom of the bottle, repeatedly blowing and beating with a microsyringe pipettor, standing at room temperature, centrifuging, and taking the supernatant for later use.

2) Total RNA extraction: adding chloroform with proper volume into the collected cell or tissue supernatant, and fully mixing until the solution is milky white; standing at room temperature and centrifuging. And (3) sucking the supernatant into a new EP tube, adding 1/2 volumes of isopropanol, fully and uniformly mixing by turning upside down, standing at room temperature, centrifuging, adding equal volume of precooled ethanol (75%), uniformly mixing by turning upside down, centrifuging at 4 ℃, removing the supernatant, drying the precipitate at room temperature, and adding a proper amount of a RNAase-free deionized water solution.

3) The purity of RNA was analyzed by agarose gel electrophoresis and the RNA concentration was determined spectrophotometrically.

3. QPCR specific procedure as in example 2

4. Statistical analysis

5. Results

The results are shown in figure 2, LOC100507053 is significantly lower expressed in breast cancer cells than in normal cell lines, with the difference being statistically significant (P < 0.05).

Example 4 overexpression of the LOC100507053 Gene

1. Cell culture

Human breast cancer cell line MCF-7 in DMEM medium containing 10% FBS and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

The cells in the culture flask were digested with pancreatin and seeded in 6-well plates to ensure that the number of cells was 2-8X 10⁵Per well, cell culture medium was added. The cell density was observed overnight the next day, and transfection was possible at cell densities above 70%.

2. Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the cDNA sequence of LOC100507053, and HindIII and XhoI restriction sites were added to the 5 'and 3' primers, respectively. cDNA obtained by blood extraction and reverse transcription of a patient with lung adenocarcinoma is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by restriction enzymes HindIII and XhoI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.

3. Transfection

The experiment was divided into three groups: the control group (MCF-7), the negative control group (pcDNA3.1-NC) and the experimental group (pcDNA3.1-1) were transfected with the vector using the liposome 3000, and the specific transfection method was performed as indicated in the specification. The transfection concentrations of the pcDNA3.1 empty vector and pcDNA3.1-1 were 0.5. mu.g/ml.

4. QPCR detection of transcription level of LOC100507053 Gene

1) The specific steps for extracting total RNA from cells were the same as in example 3

2) Specific steps of QPCR amplification are the same as in example 2

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± standard deviation, statistically analyzed using SPSS18.0 statistical software, and the difference between the LOC100507053 gene test group and the control group was determined to be statistically significant when P <0.05 using a t-test.

6. Results

The results are shown in fig. 3, where LOC100507053 expression level was significantly increased in the experimental group compared to the control group, with the difference being statistically significant (P < 0.05).

Example 5 Effect of LOC100507053 Gene on Breast cancer cell proliferation

CCK-8 experiment is adopted to detect the influence of LOC100507053 gene on the proliferation capacity of breast cancer cells.

1. Cell culture and transfection procedures were as in example 4, and the medium was changed 6h after transfection and placed in a cell incubator overnight.

2. Taking out the cells the next day, observing the growth condition of the cells under a microscope, adding pancreatin containing EDTA into 1 ml/hole, digesting the cells, removing the pancreatin after digestion is finished, adding a cell culture medium, uniformly mixing to suspend the cells, and then counting the cells.

3. The cell suspension was diluted to a concentration of 15000 cells/ml, and then seeded in a 96-well plate, 200. mu.l of the cell suspension was added to each well, and the number of cells was controlled to about 3000, and 8 wells were seeded. The pcDNA3.1-1 experimental group and the pcDNA3.1-NC control group were set. A total of 4 96 well plates were plated for 24h, 48h, 72h, 96h4 test time points, respectively.

4. And after 24h, taking out the first 96-well plate, adding 10 mu l of CCK-8 detection solution into each well, continuously putting the 96-well plate into a cell culture box, incubating for about 4h, detecting the absorbance value of each well at the wavelength of 450nm by using an enzyme-labeling instrument, and recording data.

5. And (5) repeating the operation in the step (4) after 48h, 72h and 96h respectively, and finally counting the absorbance values of all time points to make a growth curve graph.

6. Statistical analysis

The experiments were performed in 3 replicates using SPSS18.0 statistical software for statistical analysis, and the differences between the two were considered statistically significant when P <0.05 using the t-test.

7. Results

As shown in FIG. 4, compared with the control, the experimental group has obviously inhibited cell proliferation after being transfected with pcDNA3.1-1, and the statistical significance of the difference (P <0.05) indicates that LOC100507053 has the effect of inhibiting cell proliferation.

Example 6 cell migration and invasion assay

1. Transwell cell preparation

The Matrigel was thawed in an ice bath under sterile conditions, diluted 20-fold with PBS and applied to a polycarbonate membrane in a Transwell chamber at a volume of 50. mu.l/well. Standing at 37 deg.C for 4 hr, taking out after Matrigel gel polymerizes into gel, and sucking out supernatant liquid gently. 50 μ l of serum-free BSA-containing culture medium was added to each well to hydrate the basement membrane, and the membrane was left at 37 ℃ for 30 min.

2. Preparing a cell suspension

Starving the cells for 12-24h, digesting the cells, centrifuging after digestion is stopped, and removing the upper culture solution. The pelleted cells were washed with PBS and resuspended by adding serum-free medium containing BSA. Adjusting the cell density to 5 xl 0⁵One per ml.

3. Cell seeding

Mu.l of the cell suspension (100. mu.l for migration experiments and 200. mu.l for invasion experiments) was taken and added to a Transwell chamber, and 500. mu.l of DMEM medium containing FBS was added to the lower chamber of a 24-well plate. The cells were placed in a cell incubator for 24 h.

4. Dyeing process

Cells were stained with DAPI after the end of the culture. The cell of the chamber is rinsed 2 times with PBS and then placed in DAPI working solution for staining for 5-20min at room temperature. Rinsed 2 times with PBS, placed under a fluorescent microscope for observation and counted.

5. Results

The results are shown in fig. 5, compared with the control group, the migration and invasion abilities of the experimental group are obviously reduced, and the results show that the increase of the expression level of LOC100507053 can inhibit the migration and invasion of the breast cancer.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Beijing, the deep biometric information technology GmbH

<120> lncRNA related to breast cancer and application thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ttagagaagg acaagaatag 20

<210>2

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

caatatgaac agacaacag 19

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aatcccatca ccatcttcca g 21

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gagccccagc cttctccat 19

Claims

1. A method for screening potential agents for treating breast cancer in vitro, comprising:

treating a system expressing or containing the LOC100507053 gene with a candidate substance; and

detecting the expression of the LOC100507053 gene in said system;

wherein, if the candidate substance can promote the expression or activity of the LOC100507053 gene, the candidate substance is a potential substance for preventing or treating the breast cancer.

2. Use according to any one of the following:

a. the application of a reagent in preparing a product for diagnosing breast cancer is characterized in that the reagent can detect the expression level of LOC100507053 gene;

b. the application of the kit in the preparation of products for diagnosing breast cancer is characterized in that the kit comprises a reagent capable of detecting the expression level of LOC100507053 gene;

c. the application of the chip in preparing a product for diagnosing breast cancer is characterized in that the chip comprises a reagent capable of detecting the expression level of LOC100507053 gene;

d. the use of a pharmaceutical composition in the manufacture of a product for the treatment of breast cancer, wherein the pharmaceutical composition comprises an enhancer of LOC 100507053;

e. the use of a pharmaceutical composition in the manufacture of a product for the treatment of breast cancer invasion, wherein the pharmaceutical composition comprises an accelerant of LOC 100507053;

f. the application of a pharmaceutical composition in preparing a product for treating breast cancer metastasis is characterized in that the pharmaceutical composition comprises an accelerant of LOC 100507053;

g. application of LOC100507053 in vitro screening of potential substances for treating breast cancer.

3. The use of claim 2, wherein the reagents of a, b, and c comprise:

a probe specifically recognizing LOC 100507053; or

Primers for specifically amplifying LOC 100507053.

4. The use according to claim 3, wherein the primer sequence for specific amplification LOC100507053 is shown in SEQ ID No. 1-2.

5. The use according to claim 2, wherein said promoter in d, e, f is an expression vector comprising LOC 100507053.

6. The use of claim 2, wherein the pharmaceutical composition further comprises other drugs compatible with the enhancer and pharmaceutically acceptable carriers and/or excipients.