CN108192978B

CN108192978B - Tumor-related biomarker and application thereof

Info

Publication number: CN108192978B
Application number: CN201810268765.1A
Authority: CN
Inventors: 任静; 石小峰; 马翠
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2020-10-27
Anticipated expiration: 2038-03-29
Also published as: CN108192978A

Abstract

The invention discloses a tumor-related biomarker and application thereof, wherein the biomarker is LINC01336, the invention discloses a reagent, a kit and a chip for detecting the level of LINC01336, the invention also discloses a composition containing a LINC01336 promoter, and the invention also discloses application of the reagent, the kit and the chip for detecting the level of LINC01336 in preparation of a product for diagnosing gastric cancer and application of the composition containing the LINC01336 promoter in preparation of a medicine for treating gastric cancer.

Description

Tumor-related biomarker and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to a tumor-related biomarker and application thereof, and particularly relates to a gastric cancer-related biomarker LINC01336 and application thereof.

Background

Gastric cancer, the fourth largest cancer worldwide, poses a serious threat to human health, particularly in developing countries. There are nearly millions of new cases of stomach cancer worldwide each year, with developing countries accounting for nearly 2/3, 42% of which occur in china. Early diagnosis and treatment of gastric cancer are important for the illness state and prognosis of patients, and are also the main research direction to be broken through in the field of gastric cancer in China. The occurrence and development of the gastric cancer involve multigene abnormal regulation and multi-step participation, and finally, the cell biological behavior is changed from normal to abnormal. Researches show that the generation and development of the gastric cancer are closely related to genes, the correlation ratio of certain specific genes in the gastric cancer body and the genes of normal people is changed, and the possibly changed genes are the key factors for causing the gastric cancer. When the cellular behavior abnormality is based on the change of gene level, irreversible change of cellular structure and function can be caused, and the cellular canceration is possibly caused. The distant metastasis and recurrence of gastric cancer are also the leading causes of death in gastric cancer patients, and are key factors affecting clinical efficacy and prognosis. The intensive research on the pathogenesis of the gastric cancer and the specific molecular mechanism causing the gastric cancer to generate metastasis can provide a solid theoretical basis for exploring a new diagnosis method and a targeted treatment site and also provide a basis for early diagnosis and prognosis evaluation of the gastric cancer.

Studies have shown that long non-coding RNAs considered by scholars to be "noise" in human genome sequences play an important role in the development of tumors for a long time (CN201710522240.1, CN201710522694.9, CN201710522693.4), can participate in epigenetics, gene transcription and regulation of post-transcriptional levels through various pathways, and have potential as early disease diagnostic markers. However, the research on lncRNA in gastric cancer still stays at the initial stage, and the research and discovery of new lncRNA and a specific regulation mechanism thereof can provide a new theoretical basis for the research on pathogenesis of gastric cancer and provide a new way for clinical diagnosis and treatment of gastric cancer.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide lncRNA related to the occurrence and development of gastric cancer, thereby providing a new means for early diagnosis of gastric cancer and treatment of gastric cancer, invasion and metastasis of gastric cancer.

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

in a first aspect of the invention, an agent is provided, which is capable of detecting the expression level of the LINC01336 gene.

Further, the agent is selected from:

a probe that specifically recognizes LINC 01336; or

Primers specifically amplifying LINC 01336.

Further, the primer sequence of the specific amplification LINC01336 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, a composition is provided, said composition comprising an accelerator of LINC 01336.

Further, the promoter is an expression vector containing LINC 01336.

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

A fifth aspect of the present invention provides a method of screening a drug for preventing or treating gastric cancer, the method comprising:

treating a system expressing or containing the LINC01336 gene with a substance to be screened; and

detecting the expression of the LINC01336 gene in the system;

wherein, if the substance to be screened can promote the expression or activity of the LINC01336 gene, the substance to be screened is indicated to be a medicine for treating gastric cancer.

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

a. the use of an agent according to the first aspect of the invention in the manufacture of a product for the diagnosis of gastric cancer;

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

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

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

e. the application of the pharmaceutical composition of the fourth aspect of the invention in preparing products for treating gastric cancer invasion;

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

g. the method of the fifth aspect of the present invention is used for screening potential substances for treating gastric cancer.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC01336 gene in gastric cancer tissue by QPCR;

FIG. 2 is a graph showing the detection of the expression of LINC01336 gene in a gastric cancer cell line by QPCR;

FIG. 3 is a graph showing the detection of the transfection of LINC01336 in gastric cancer cells by QPCR;

FIG. 4 is a graph showing the effect of LINC01336 gene on gastric cancer cell proliferation measured by the CCK-8 method;

FIG. 5 is a graph of the effect of LINC01336 on gastric cancer cell migration and invasion measured using a Transwell chamber; wherein

Panel a is a graph of the effect of LINC01336 on gastric cancer cell migration; panel B is a graph of the effect of LINC01336 on gastric cancer cell invasion.

Detailed Description

According to the invention, through extensive and intensive research, the expression of lncRNA in a gastric cancer specimen in tumor tissues and tissues beside the cancer is detected by adopting an lncRNA chip which covers the most wide database 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 gastric cancer is discussed, so that a better way and a better method are found for the diagnosis and the targeted therapy of the gastric cancer. Through screening, the invention discovers that LINC01336 in gastric cancer is remarkably reduced. Experiments prove that the proliferation, migration and invasion of gastric cancer cells can be effectively inhibited by improving the expression level of LINC 01336.

LINC01336 gene

LINC01336 is located in the 3-band of region 1 of human staining long arm No. 5, and the nucleotide sequence of a representative human LINC01336 gene is shown in LINC01336 gene (NR _126375.1) in GeneBank, the current International public nucleic acid database. The full-length LINC01336 nucleotide sequence or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. LINC01336 in the present invention includes wild type, mutant type or fragments 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 referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein 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.beta.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 invention provides products for detecting the expression level of LINC01336 gene in a sample, and the products comprise (but are not limited to) preparations, chips or kits. 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 in LINC 01336.

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 LINC 01336.

In a preferred embodiment, the preparation or the kit further comprises a marker for labeling the RNA sample, and a substrate corresponding to the marker.

As a more preferred embodiment, the kit may further comprise 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.

As a further preferred embodiment, the kit further comprises instructions for use and/or chip image analysis software.

Accelerator and composition

Based on the findings of the present invention, the present invention provides a composition comprising an accelerator of LINC 01336.

The promoter for LINC01336 refers to any substance which can improve the stability of LINC01336 gene or an expression product, up-regulate the expression of LINC01336, prolong the effective action time of lncRNA LINC01336 or promote the transcription of LINC01336 gene, and the substances can be used for the promoter, can be used as substances which are useful for up-regulating the expression of the LINC01336 gene, and can be used for preventing or treating lung adenocarcinoma.

In a preferred mode of the invention, the LINC01336 promoter is an expression vector containing LINC 01336. 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.

The medicine and/or composition comprises the accelerating agent of LINC01336, and/or other medicines compatible with the accelerating agent, and a pharmaceutically acceptable carrier and/or auxiliary material.

The pharmaceutically acceptable carrier may be one or more and includes, but is not limited to, binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants and/or time delay agents (timedelayagent). Suitable binders include acacia, gelatin, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methyl cellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint oil, oil of wintergreen, cherry, citrus or raspberry flavors. Suitable coating agents include polymers or copolymers of acrylic and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delays include glyceryl monostearate or glyceryl distearate. 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 sufficient to treat the disease, at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined according to the type of the subject, the severity of the disease, the age and sex of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the treatment time, the drug to be used in combination with the composition, and other known factors in the medical field. The pharmaceutical compositions of the present invention may be used alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. The compositions may be administered in one or more dosage forms. In view of all the above factors, it is important to administer the composition at the minimum amount capable of exhibiting the maximum effect without causing side effects, which can be readily determined by one skilled in the art.

The term "effective" means that the treatment results in a decrease in pathologically up-regulated gene expression, an increase in pathologically down-regulated gene expression or a decrease in the size, prevalence (prevalence) or metastatic potential of gastric cancer tissue in the subject. The term "effective" when used prophylactically to refer to treatment that delays or prevents the formation of gastric tumors, or delays, prevents or alleviates the clinical symptoms of gastric cancer. Evaluation of gastric tumors can be performed according to standard clinical protocols.

The medicament or composition of the present invention may further comprise one or more anticancer agents. In a specific embodiment, the medicament or composition comprises at least one compound inhibiting the expression of the LINC01336 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 medicament or composition of the invention can also be used in combination with other medicaments for the treatment of gastric cancer, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

The medicaments or compositions of the invention may also be administered separately to other therapeutic compounds in a single composition or in a different dosage form than the main 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 treating gastric cancer, which comprises the following steps:

in an experimental group, adding a compound to be tested into a culture system, and determining the expression level of LINC 01336; in a control group, no test compound is added into the same culture system, and the expression level of LINC01336 is determined; wherein if the expression level of LINC01336 in the experimental group is greater than that in the control group, the substance to be screened is the drug of LINC 01336.

In the present invention, the method further comprises: the drug obtained in the above step is further tested for its gastric cancer inhibitory effect, and if the test compound has a significant gastric cancer inhibitory effect, the compound is indicated as a drug for preventing or treating gastric 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.

When a compound isolated by the screening method of the present invention is administered as a drug to a human or other mammal, including but not limited to mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees, the isolated compound can be administered directly or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug may be orally administered as sugar-coated tablets, capsules, elixirs and microcapsules, as desired; or as a sterile solution or suspension in water or any other pharmaceutically acceptable liquid for parenteral administration in the form of an injection. For example, the compounds may be mixed together in unit dosage forms (unit dose) as required for generally accepted modes of pharmaceutical administration, with pharmaceutically acceptable carriers or media including, but not limited to, sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients (exipient), vehicles (vehicle), preservatives, binders and the like. Depending on the content of the active ingredient in these preparations, an appropriate amount to be administered within a prescribed range can be obtained.

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with gastric cancer

1. Sample collection

Samples of tissue adjacent to and including gastric cancer were collected in 8 cases, 3 normal gastric tissues were collected, all cases received no chemotherapy or radiotherapy prior to surgery, all patients had known consent, and all had given consent from the tissue ethics committee.

2. Preparation of RNA samples

Tissue RNA extraction was performed using a tissue RNA extraction kit from QIAGEN, and the procedures were performed according to the specific procedures described in the specification.

3. Total RNA quantitation and purity analysis

The optical density values of total RNA at 280nm and 260nm, as OD, were determined using a Bio-Red UV spectrophotometer₂₆₀/OD₂₈₀The value of (A) is 1.8 to 2.0, and the purity of the total RNA is considered to be reliable, and the total RNA is used for the next experiment.

4. lncRNA expression chip analysis

The difference in lncRNA expression profiles between gastric cancer tissues and paracarcinoma tissues was examined using Arraystar Human 1ncRNA Array from Arraystar.

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 LINC01336 exhibits differential expression in gastric cancer patients, with expression levels significantly higher in the paracancerous tissues compared to the paracancerous tissues as well as normal stomach tissues, approximately 6.4 times higher than in the cancerous tissues.

Example 2 QPCR sequencing validation of differential expression of LINC01336 Gene

1. Large sample QPCR validation was performed on the differential expression of LINC01336 gene. 50 cases of the tissues adjacent to gastric cancer and the stomach cancer and 10 cases of the normal stomach tissue were selected in the same manner as in example 1.

2. RNA extraction

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

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 LINC01336 gene and GAPDH gene in Genebank and were synthesized by Bomader Biotech. The specific primer sequences are as follows:

LINC01336 gene:

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

the reverse primer was 5'-GTAGGTAAAGCAGAGAGAAAT-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

And analyzing the working characteristics of the testees of the LINC01336 by using a pROC package in the R language, calculating a binomial accurate confidence space, and drawing an ROC curve.

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 tissues beside the gastric cancer and the normal stomach tissue, the expression of LINC01336 in the gastric cancer tissue is reduced, the difference has statistical significance (P is less than 0.05), and the result is consistent with the chip detection result; the ROC curve result shows that the AUC value of LINC01336 is as high as 0.901, and the LINC01336 has high specificity and sensitivity, which indicates that LINC01336 can be applied to specific diagnosis of gastric cancer, and LINC01336 has high accuracy in diagnosis of gastric cancer.

Example 3 expression of LINC01336 in gastric cancer cell lines

1. Cell culture

Human immortalized gastric mucosal epithelial cell lines GES-1, human gastric cancer cell lines HGC-27, MGC-803, AGS (all available from Laidel Biotech Co., Ltd., Guangzhou) were cultured in RPMI1640 containing 10% fetal bovine serum and 1% P/S at 37 ℃ with 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days, passaged by conventional digestion with 0.25% EDTA-containing trypsin, and cells in logarithmic growth phase were taken for experiment.

2. Extraction and concentration determination of RNA

Total cellular RNA was extracted using a QIAGEN's cellular RNA extraction kit, and the specific procedures were as described in the instruction manual.

3. QPCR specific procedure as in example 2

4. Statistical analysis

5. Results

The results are shown in fig. 2, LINC01336 is significantly lower in expression in gastric cancer cell lines than in normal cell lines (gastric mucosal epithelial cell lines), the difference is statistically significant (P <0.05), HGC-27 cells are selected for subsequent experiments to study the effect of LINC01336 on gastric cancer cells.

Example 4 overexpression of LINC01336 Gene

1. Cell culture

The human gastric cancer cell line HGC-27 was cultured in the same manner as in example 3.

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

Synthesizing a specific PCR amplification primer according to the cDNA sequence of LINC01336, respectively adding restriction sites to a 5 'end primer and a 3' end primer, inserting the primers into a eukaryotic expression vector pcDNA3.1 after restriction, and connecting the obtained recombinant vector pcDNA3.1-1 for subsequent experiments.

3. Transfection

The experiment was divided into three groups: the control group (HGC-27), 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 instruction

4. QPCR detection of transcript level of LINC01336 gene

1) Extraction of Total RNA from cells

Total cellular RNA was extracted using QIAGEN's cellular RNA extraction kit, and the detailed procedures were as follows.

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 ± sd, statistically analyzed using SPSS18.0 statistical software, and the differences between the experimental and control LINC01336 gene groups were considered statistically significant when P <0.05 using the t-test.

6. Results

The results are shown in fig. 3, where LINC01336 expression level in the experimental group was significantly increased compared to the control group, with a statistical significance of the difference (P < 0.05).

Example 5 Effect of LINC01336 Gene on gastric cancer cell proliferation

MTS experiment is adopted to detect the influence of LINC01336 gene on the proliferation capacity of gastric cancer cells.

1. The cell culture and transfection procedures were the same as in example 4.

2. Cells treated in each group were trypsinized, resuspended, counted, and adjusted to a cell concentration of lx 10⁵The cells were seeded in 96-well plates at a density of 100. mu.l/well, i.e.1X 10 cells per well⁴And (4) respectively.

3. After the cells reach corresponding detection time points (0d, 24h, 48h, 72h and 96h), adding a Celititer96AQ single-solution cell proliferation detection (MTS) reagent according to 10 mu L/hole, and oscillating for 1-2 min by a micro oscillator; placing in 5% CO₂Incubate at 37 ℃ for 4 h.

4. The plate was read by a microplate reader and the absorbance (A) was measured at 490 nm.

5. 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.

6. Results

As shown in FIG. 4, compared with the control, the experimental group has the advantages that after pcDNA3.1-1 is transfected, the cell proliferation is obviously inhibited, the difference has statistical significance (P <0.05), the LINC01336 has the effect of inhibiting the cell proliferation, and the increase of the level of the LINC01336 can inhibit the proliferation of the gastric cancer cell.

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 and centrifuging with pancreatin, resuspending in serum-free medium, adjusting the cell density to 5 × l0⁵One per ml.

3. Cell seeding

The cell suspension was taken in an amount of 200. mu.l to a Transwell chamber, and 500. mu.l of DMEM medium containing FBS was added to the lower chamber of the 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 level of LINC01336 can inhibit the migration and invasion of the gastric cancer, and suggest that LINC01336 can be used as a target for the treatment of the migration and invasion of the gastric 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

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<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gagccccagc cttctccat 19

Claims

1. A method of screening for a drug for the treatment of gastric cancer, the method comprising:

detecting the expression of the LINC01336 gene in the system;

wherein, if the substance to be screened can promote the expression of the LINC01336 gene, the substance to be screened is a medicine for treating gastric cancer.

2. Use according to any one of the following:

a. the application of the reagent in preparing a product for diagnosing gastric cancer is characterized in that the reagent can detect the expression level of LINC01336 gene;

b. the application of the kit in preparing a product for diagnosing gastric cancer is characterized in that the kit comprises a reagent capable of detecting the expression level of LINC01336 gene;

c. use of a chip for the preparation of a product for diagnosing gastric cancer, wherein the chip comprises a reagent capable of detecting the expression level of LINC01336 gene;

d. use of a composition for the manufacture of a medicament for the treatment of gastric cancer, wherein the composition comprises a promoter for LINC01336, said promoter promoting the expression level of LINC 01336;

g. use of the method of claim 1 for screening a medicament for the treatment of gastric cancer.

3. The use of claim 2, wherein the agents in a-c comprise:

a probe that specifically recognizes LINC 01336; or

Primers specifically amplifying LINC 01336.

4. The use according to claim 3, wherein the primer sequence for specific amplification of LINC01336 is shown as SEQ ID No. 1-2.

5. The use of claim 2, wherein said promoter in d is an expression vector comprising LINC 01336.

6. The use of claim 5, wherein the composition further comprises other drugs compatible with the enhancer and pharmaceutically acceptable excipients.