CN113373229B - Gastric cancer related biomarker and application thereof - Google Patents

Abstract

The invention discloses a gastric cancer-related biological marker and its application. The biological marker is LINC01895. In the specific embodiment of the present invention, the expression of LINC01895 is up-regulated in the gastric cancer cells in which the Notch1 gene is silenced, and the expression of LINC01895 in gastric cancer tissue is further detected by qPCR. The results show that the expression of LINC01895 in gastric cancer tissue is down-regulated, and has better Diagnostic efficacy, the experimental results suggest that LINC01895 can be used to diagnose gastric cancer or predict the prognosis of gastric cancer patients using Notch1 inhibitor. The present invention also provides a method for screening candidate drugs for preventing or treating gastric cancer.

Description

Gastric cancer-related biomarkers and their applications

technical field

The invention belongs to the field of biomedicine, and particularly relates to gastric cancer-related biological markers and applications thereof.

Background technique

Gastric cancer is one of the common malignant tumors that seriously endanger human health, and its incidence ranks fourth among the most common malignant tumors in the world, second only to lung cancer, prostate cancer and colon cancer (Patru C L, Surlin V, Georgescu I, et al. Current. issues in gastric cancer epidemiology.[J].rev med chir soc med natiasi,2013,117(1):199-204.). According to statistics, the expected incidence of gastric cancer is 640,000 people. Among women, the incidence of gastric cancer ranks fifth after breast cancer, colon cancer, cervical cancer and lung cancer, with an estimated annual incidence of 350,000. The mortality rate of gastric cancer accounts for 8-10% of the global annual mortality rate of malignant tumors, and the mortality rate of gastric cancer is as high as 70%, which is significantly higher than that of other malignant tumors such as prostate cancer (30%) and breast cancer (33%).

Currently, the diagnosis of gastric cancer relies on endoscopy, pathological examination and the determination of tumor markers [including carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), cancer antigen 72-4 (CA72-4) and Cancer Antigen 50 (CA50), etc.]. However, although endoscopy and pathological examination are the "gold standard" for diagnosing gastric cancer, they cannot be used for early cancer screening due to their high cost, difficulty in obtaining materials, and pain for patients. Tumor markers are also of limited utility due to the lack of high specificity and sensitivity. Therefore, to optimize treatment strategies, predict efficacy, and prolong patient survival, novel diagnostic markers must be identified as soon as possible. Circulating long non-coding RNAs (lncRNAs) can be directly detected from patient plasma and serum, and have the advantages of convenient and rapid detection, non-invasiveness, high sensitivity and specificity, and are regarded as potential tumor markers for gastric cancer.

Notch is an important signaling receptor molecule, which determines the time and space of species development in vertebrates and invertebrates, and determines the final differentiation of various cells. Notch is a single-pass transmembrane protein with two well-defined extracellular domains: an epidermal growth factor-like repeat, whose primary function is to bind ligands; and three cysteine-rich Notch/lin-12 repeats , whose main function is to block signal transduction in the absence of ligands. The cytoplasmic portion of Notch includes a regulatory amino acid metabolism domain, six typoin repeat domains, two nuclear localization signal sequence domains, a transcriptional activation domain, and a prohelic acid-glutamic acid-serine-threonine-rich domain set sequence. There are four members of the Nocth family, namely Notch1-4, which bind to the corresponding ligands to release the intracellular segment and translocate into the nucleus to bind to downstream target gene sites to regulate gene transcription. Studies have shown that the Notch family is involved in the occurrence and development of various tumors, and silencing the expression of Notch1 can inhibit the proliferation and invasion of gastric cancer cell lines (Wei G, Chang Y, Zheng J, et al. Notch1 silencing inhibits proliferation and invasion in SGC-7901 gastric cancer. cells[J]. Molecular Medicine Reports, 2014, 9(4): 1153-1158.), therefore, searching for differentially expressed lncRNAs by silencing the expression of Notch1 is expected to find new biomarkers for gastric cancer.

SUMMARY OF THE INVENTION

In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a biomarker that can be used to diagnose gastric cancer, and to provide a new method for the diagnosis of gastric cancer. In order to achieve this purpose, the present invention adopts the following technical solutions:

The first aspect of the present invention provides the application of a reagent for detecting the expression level of LINC01895 in a sample in preparing a product for diagnosing gastric cancer or predicting the prognosis of gastric cancer patients using Notch1 inhibitor.

The "LINC01895" refers to the gene whose Gene ID is 105371964.

Further, the reagents include reagents for detecting the expression level of LINC01895 in the sample through sequencing technology, nucleic acid hybridization technology, and nucleic acid amplification technology.

Non-limiting examples of nucleic acid sequencing methods of the present invention include next-generation sequencing (deep sequencing/high-throughput sequencing), which is a single-molecule cluster-based sequencing-by-synthesis technology based on proprietary reversible termination Principles of chemical reactions. During sequencing, random fragments of genomic DNA are attached to an optically transparent glass surface. After extension and bridge amplification, these DNA fragments form hundreds of millions of clusters on the glass surface, each cluster has thousands of copies of the same template Then, using four special deoxyribonucleotides with fluorescent groups, the template DNA to be tested is sequenced by reversible sequencing-by-synthesis technology.

Nucleic acid hybridization methods in the present invention include, but are not limited to, in situ hybridization (ISH), microarrays, and Southern or Northern blotting. In situ hybridization (ISH) is a method that uses labeled complementary DNA or RNA strands as probes to localize a section or section of tissue (in situ) or, if the tissue is small enough, whole tissue (whole tissue-embedded ISH) for specificity Hybridization of DNA or RNA sequences. DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNAs and other transcripts (eg, ncRNAs) within tissue sections or whole tissue embeddings. Sample cells and tissues are typically processed to immobilize target transcripts in situ and increase probe access. The probes hybridize to the target sequence at high temperature, and the excess probes are washed away. Localization and quantification of probes labeled with radioactive, fluorescent, or antigen-labeled bases in tissues are performed using autoradiography, fluorescence microscopy, or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Southern and Northern blots were used to detect specific DNA or RNA sequences, respectively. DNA or RNA extracted from the sample is fragmented, separated by electrophoresis on a Matrigel gel, and then transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. The hybridization probe bound to the filter is detected. A variation of this procedure is a reverse Northern blot, where the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from tissue and labeled.

The nucleic acid amplification technology in the present invention is selected from 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). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), and TMA and NASBA directly amplify RNA.

Typically, 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; RT-PCR uses reverse transcriptase (RT) to prepare complementary DNA (cDNA), which is then amplified by PCR to generate multiple copies of DNA; TMA autocatalytically synthesizes multiple copies of the target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein the target sequence Multiple copies of RNA autocatalytically generate additional copies, TMA optionally includes the use of blocking, moieties, termination moieties and other modified moieties to improve the sensitivity and accuracy of the TMA process; LCR uses regions adjacent to the target nucleic acid Two sets of complementary DNA oligonucleotides that hybridize. DNA oligonucleotides are covalently ligated by DNA ligase in repeated cycles of heat denaturation, hybridization, and ligation to produce detectable double-stranded ligated oligonucleotide products; SDA uses multiple cycles of the following steps: primers Sequence pair annealing to the opposite strand of the target sequence, primer extension in the presence of dNTPαS to generate a double-stranded hemiphosphorothioated primer extension product, endonuclease for hemi-modified restriction endonuclease recognition sites Dicer-mediated nicking, and polymerase-mediated extension from the 3' end of the nick to displace existing strands and generate strands for the next round of primer annealing, nicking, and strand displacement, resulting in the production of geometric amplification.

The terms "sample" and "sample" are used interchangeably herein to refer to a composition obtained or derived from a subject (eg, an individual of interest) that contains and/or physiological characteristics to characterize and/or identify cells and/or other molecular entities. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (eg, tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicles Fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluids, tissue extracts such as homogenized Tissue, tumor tissue, cell extracts, and combinations thereof.

As a preferred embodiment, the sample is selected from tissue.

Further, the reagent is selected from:

A probe that specifically recognizes LINC01895;

Or primers that specifically amplify LINC01895.

"Probe" refers to a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe 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 target polynucleotides that lack complete sequence complementarity to the probe. Probes can be directly or indirectly labeled. Hybridization means, including, but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

Further, the primer sequences for specific amplification of LINC01895 are shown in SEQ ID NOs. 5-6.

The second aspect of the present invention provides a product for diagnosing gastric cancer or predicting the prognosis of gastric cancer patients using a Notch1 inhibitor, the product comprising a reagent for detecting the expression level of LINC01895 in a sample.

Further, the products include chips, kits, and nucleic acid membrane strips.

In the present invention, the nucleic acid membrane strip includes a substrate and oligonucleotide probes immobilized on the substrate; the substrate can be any substrate suitable for immobilizing oligonucleotide probes, such as nylon membrane, nitrocellulose Film, polypropylene film, glass sheet, silica gel wafer, miniature magnetic beads, etc.

In the present invention, "chip", also referred to as "array", refers to a solid support comprising linked nucleic acid or peptide probes. Arrays typically contain a variety of different nucleic acid or peptide probes attached to the substrate surface at different known locations. These arrays, also referred to as "microarrays," can typically be produced using mechanical synthesis methods or light-guided synthesis methods that combine a combination of lithographic and solid-phase synthesis methods. Arrays may comprise flat surfaces, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. Arrays can be packaged in a manner that allows diagnostic or other manipulation of a fully functional device.

A "microarray" is an ordered arrangement of hybridization array elements, such as polynucleotide probes (eg, oligonucleotides) or binding agents (eg, antibodies), on a substrate. The substrate can be a solid substrate, eg, glass or silica slides, beads, fiber optic binders, or semi-solid substrates, eg, nitrocellulose membranes. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

The present invention provides a kit for diagnosing gastric cancer in a subject, and the kit is used for determining the level of a biomarker. The kit may include materials and reagents suitable for selectively detecting the presence of a biomarker or panel of biomarkers for the diagnosis of gastric cancer in a sample derived from a subject. For example, in one embodiment, the kit may include reagents that specifically hybridize to a biomarker. Such reagents may be nucleic acid molecules in a form suitable for detection of biomarkers, eg, probes or primers. The kit can include reagents for performing an assay to detect one or more biomarkers, eg, reagents that can be used to detect one or more biomarkers in a qPCR reaction. The kit can also include a microarray for detecting one or more biomarkers.

In further embodiments, the kit may contain instructions for suitable operating parameters in the form of labels or product inserts. For example, the instructions may include instructions on how to collect the sample, how to determine the level of one or more biomarkers in the sample, or how to correlate the level of one or more biomarkers in the sample with the gastric cancer status of the subject information or guidance.

Further, the kit may contain one or more containers with biomarker samples for use as reference standards, suitable controls, or calibrations for assays to detect biomarkers in test samples.

Further, the product also includes reagents for processing samples.

A third aspect of the present invention provides a composition comprising an accelerator of LINC01895.

Further, the promoter is a vector that specifically promotes the expression of LINC01895.

The fourth aspect of the present invention provides the use of the composition described in the third aspect of the present invention in preparing a pharmaceutical composition for preventing or treating gastric cancer.

The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier. The term carrier includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic, thickening or emulsifying agents, preservatives, solid viscosity agents suitable for preparing the particular dosage form desired. Mixtures, lubricants, etc. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and derivatives thereof, such as carboxymethyl cellulose Sodium plain, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil , corn oil, and soybean oil; glycols, such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline ; Ringer's solution; ethanol and phosphate buffered saline, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents may also be present in the composition at the judgment of the formulator. , release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.

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

(1) Treat a system expressing or containing LINC01895 with the substance to be tested;

(2) detecting the expression level of LINC01895 in the system;

(3) Select substances that can increase the expression level of LINC01895 as candidate drugs.

Advantages and beneficial effects of the present invention:

The present invention discovers for the first time that gastric cancer can be diagnosed or the prognosis of gastric cancer patients using Notch1 inhibitor can be predicted by detecting the expression level of LINC01895.

The invention also discloses a method for screening candidate drugs for preventing or treating gastric cancer.

Description of drawings

Fig. 1 is a graph showing the expression level of Notch1 protein in gastric cancer cells after transfection with Notch1 knockout lentivirus; wherein, Fig. A is a graph showing the expression level of Notch1 protein in MKN45 cells; Fig. B is a graph showing the expression level of Notch1 protein in MGC-803 cells; Fig. C is the Notch1 protein expression level map of AGS cells;

Figure 2 is a statistical graph of the relative expression of LINC01895 in gastric cancer tissue detected by qPCR;

Figure 3 is a ROC curve diagram of LINC01895 in the diagnosis of gastric cancer.

Detailed ways

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

Example 1 Interfering with Notch1 gene expression in gastric cancer cells

1. Experimental materials

1. Experimental reagents: as shown in Table 1, and antibody information as shown in Table 3;

2. Experimental equipment: as shown in Table 2;

3. Notch1 knockout lentivirus, negative control lentivirus;

4. Gastric cancer cell lines MKN45, MGC-803, AGS.

Table 1 Experimental reagents

Table 2. Experimental Apparatus

Table 3 Antibody Information

2. Experimental method

1. Virus infection

(1) Plate: After the cells in the logarithmic growth phase are digested and plated, 1*10 ⁵ /well is inoculated into a 12-well plate and grown overnight;

(2) Infection: suck out the culture solution in the 12-well plate, replace with fresh culture solution, and add the virus solution diluted by PBS concentration gradient at the same time, and put it into the incubator after mixing evenly;

(3) The liquid can be changed around 24h, and the fluorescence can be seen around 48h.

2. Western Blot Verification

(1) Total cell protein extraction

a. Rinse cells 2-3 times with PBS. One final time, the residual liquid was completely sucked dry.

b. Add an appropriate volume of RIPA (add protease inhibitor within a few minutes before use) to the culture plate and bottle for 3-5min. During this period, shake the culture plate and bottle repeatedly to make the reagent fully contact with the cells.

c. Scrape off the cells and reagents with a cell scraper and collect them into a 1.5mL centrifuge tube.

d. Ice bath for 30 minutes, during which time, pipette repeatedly to ensure complete cell lysis.

e. 12000rpm, 4℃, centrifuge for 10min, collect the supernatant, which is the total protein solution.

(2) Determination of protein concentration

Take the undenatured protein solution and measure the protein concentration with BCA protein concentration assay kit.

(3) Protein denaturation

The protein solution was added to 5* protein loading buffer at a ratio of 4:1, denatured in a boiling water bath for 15 min, and stored in a -20°C refrigerator for later use.

(4) SDS-PAGE electrophoresis

a. Configure the protein gel

b. Put the gel maker into the electrophoresis tank and add enough electrophoresis solution to load the sample for electrophoresis. The sample is added to the electrophoresis wells and electrophoresed. The stacking gel voltage is 75V, and the separating gel is 120V.

c. The electrophoresis can be terminated until the bromophenol blue has just run out, and the membrane is transferred.

(5) Transfer film

a. Prepare 6 pieces of 7×9cm filter paper and a PVDF membrane of moderate size. The PVDF membrane should be activated with methanol before use.

b. Put the clip for transferring membrane, two sponge pads, one glass rod, filter paper and activated PVDF membrane into the basin with transfer solution.

c. Open the clip to keep the black side level. Put a sponge and three layers of filter paper on the pad.

d. Carefully peel off the separation glue and place it on the filter paper, and cover the PVDF membrane on the glue without air bubbles. Cover the membrane with three filter papers and remove air bubbles. Finally cover with another sponge pad.

e. Transfer conditions (wet transfer)

Fast rotation: 300mA constant current transfer film for half an hour, or 200mA transfer film for 1 hour, the time can be slightly adjusted, and the current is adjusted accordingly. During the transfer process, place the transfer tank in ice water to cool down.

Slow transfer: transfer the membrane overnight, and transfer the membrane overnight at 25V constant voltage.

(6) Immune response

a. The transferred membrane was blocked with 5% skim milk (0.5% TBST) on a destaining shaker at room temperature for 1 h.

b. Dilute the primary antibody (5% nonfat milk in TBST, 5% BSA in TBST for phosphorylated protein) and incubate at 4°C overnight (fast rotation), or incubate the antibody at 4°C for 3 hours (slow rotation).

c. Wash three times with TBST on a destaining shaker at room temperature, 5 min each time.

d. Dilute the secondary antibody by 3000 times with TBST, incubate at room temperature for 30 minutes, and wash three times with TBST on a destaining shaker at room temperature for 5 minutes each time.

(7) Chemiluminescence

Mix the two reagents, ECLA and ECLB, in a centrifuge tube in an equal volume in a dark room, put double gloves or other transparent films on the exposure box, put the protein side of the PVDF membrane between the two layers of the exposure box, and add and mix The good ECL solution is fully reacted. After 1-2 minutes, the residual liquid is removed, and the upper layer of film is covered to start exposure. The exposed film is developed and fixed with developing and fixing reagents. Adjust exposure conditions according to different luminous intensities.

(8) Gel image analysis

The film is scanned and archived, PhotoShop organizes and decolorizes, and the Alpha software processing system analyzes the optical density value of the target band.

3. Experimental results

As shown in Figure 1, after transfection of Notch1 knockout lentivirus for interference, the expression of Notch1 protein in gastric cancer cells (MKN45, MGC-803, AGS) was significantly down-regulated, which proved that the gastric cancer cell line with knockdown of Notch1 gene was successfully obtained.

Example 2 Screening of differentially expressed genes

1. High-throughput sequencing data processing

The blank control and Notch1 gene knockdown group gastric cancer cells in Example 1 were collected by Trizol method, each with 3 replicates, and subjected to high-throughput sequencing, and the original sequencing data were filtered:

1. Remove the adapter sequence in the reads;

2. Remove the bases containing non-AGCT at the 5' end before cutting;

3. Trim the ends of reads with lower sequencing quality (sequencing quality value is less than Q20);

4. Remove reads with a ratio of N up to 10%;

5. Discard the adapter and the small fragments whose length is less than 25bp after quality trimming.

The data volume statistics were performed on the quality sheared sequences, and the results are shown in Table 4.

Table 4 Sequencing data statistics table

Second, the reference sequence comparison analysis

The high-quality sequencing sequences obtained after quality control were compared with the designated reference genome. The study species was human. The reference genome was from the Ensembl database, and the genome version was GRCh38.p13.

Analysis software: hisat2, version v 2.1.0, and the statistical table of the comparison results with the reference genome is shown in Table 5.

Table 5 Clean Mapping ratio statistics

3. mRNA expression analysis

In RNA-seq analysis, gene expression levels are calculated by the number of sequences (clean reads) aligned to a reference genomic region. According to the comparison results of all samples and the reference genome, the count value of each gene/transcript in the sample is calculated, and this value is used as the expression level of the gene/transcript in the sample. Finally, the differential significance analysis was performed on the expression of all genes/transcripts in each group of samples, and the relative differentially expressed genes/transcripts were found and visualized.

The software DESeq2 was used to identify differential genes from RNA-Seq data, which integrated Fisher's test and likelihood ratio test based on binomial distribution model for differential expression test. Significant difference mRNA screening conditions: P_value<0.05, |logFC|>0.585.

4. Data Analysis Results

145 differential genes were screened by the above criteria, including 102 up-regulated genes and 43 down-regulated genes. The gene LINC01895 described in the present invention is up-regulated in gastric cancer cells in the Notch1 gene knockdown group, as shown in Table 6. The results indicate that Notch1 inhibitors may inhibit the proliferation and invasion of gastric cancer cell lines by promoting the expression of LINC01895.

Table 6 Differential expression of LINC01895

Gene baseMean log2 FoldChange P.Value updown LINC01895 11.689 2.412 0.022 Up

Example 3 Detection of changes in the expression of LINC01895 in gastric cancer tissue samples by qPCR

1. Experimental materials

1. Samples: 46 gastric tissue samples, including 23 gastric cancer tissue samples and 23 paracancerous control tissues.

2. Experimental reagents: as shown in Table 7.

Table 7 List of reagents used

3. The main instruments of the experiment: as shown in Table 8.

Table 8 List of instruments used

2. Experimental method

1. Primer Design

Real Time PCR detection of target gene primers. The primers were synthesized in Biomed, and the primer sequences are shown in Table 9.

Table 9 Primer sequences

primer name Primer sequence (5'to3') GAPDH-F (internal reference) CTGGGCTACACTGAGCACC (SEQ ID NO. 1) GAPDH-R (internal reference) AAGTGGTCGTTGAGGGCAATG (SEQ ID NO. 2) ACTB-F (internal reference) GATCAAGATCATTGCTCCTCCT (SEQ ID NO. 3) ACTB-R (internal reference) TACTCCTGCTTGCTGATCCA (SEQ ID NO. 4) LINC01895-F TAAACAAGCAGCCCCTGTCT (SEQ ID NO. 5) LINC01895-R CGCAGGTAATAGGTGGCAGT (SEQ ID NO. 6)

2. Extract total RNA from the sample

(1) Add 1 mL of TRIzol to the glass homogenization bottle in the ultra-clean bench (bake the homogenization bottle in an oven at 180 degrees for 4 hours in advance), press the homogenization bottle to the instrument, and weigh 50-100 mg of tissue Put it into a glass homogenizer bottle, adjust the rotation speed to about 1500 rpm, start to homogenize in an ice-water bath, grind for 30s every 30s, stop for 30s, and repeat 3-4 times. The sample volume should not exceed 10℅ of the TRIzol volume.

(2) The TRIzol-added sample was placed at room temperature (15-30° C.) for 10 minutes to completely separate the nucleic acid-protein complexes.

(3) Add 200 μL of chloroform to 1 mL of TRIzol, shake vigorously for 2 minutes, shake twice every 1 minute, and then stand still for 7 minutes after 5-6 times.

(4) 4°C, 12000rpm, centrifugation for 15min. The sample is divided into three layers: the bottom layer is the yellow organic phase, the upper layer is the colorless aqueous phase and an intermediate layer. The RNA was mainly in the aqueous phase, and the volume of the aqueous phase was about 60℅ of the TRIzol used.

(5) Transfer the upper aqueous phase to a new EP tube (about 400 μL, try not to pipette into the middle layer to avoid contamination). Add 500 μL of isopropanol and leave at room temperature for 10 min.

(6) Prepare 75% ethanol and pre-cool in an ice box.

(7) 4 ℃, 12000rpm, centrifuge for 15min, white precipitate appears at the bottom of the tube after centrifugation. Remove the supernatant carefully with a pipette.

(8) Add 1 mL of 75℅ cold ethanol and shake to wash the precipitate. 4°C, 7500rpm, centrifuge for 5min, carefully discard the supernatant.

(9) Invert the EP tube on the filter paper to absorb excess water, and carefully aspirate the liquid in the tube with a 10 μL pipette tip (the pipette tip should not touch the RNA), and leave the EP tube at room temperature for 5 minutes (too long, too drying will cause RNA activity decreases), the RNA becomes transparent;

(10) Add 40 μL of RNase-free water (DEPC water), use naodrop to detect the OD value and concentration, and mark the tube;

(11) Store in -80°C refrigerator.

3. Reverse transcription to synthesize mRNA and cDNA

Use FastKing cDNA First Strand Synthesis Kit (Cat. No.: KR116) to perform reverse transcription of mRNA. First, remove the genomic DNA reaction, add 2.0 μL of 5×gDNA Buffer, 1 μg of TotalRNA to the test tube, add Rnase FreeddH2O to make the total volume to 10 μL, water bath Heat the pot at 42°C for 3 min, then add 2.0 μL of 10×King RT Buffer, 1.0 μL of FastKing RT Enzyme Mix, 2.0 μL of FQ-RT Primer Mix, 5.0 μL of RNase Free ddH2O, and add them to the above test tube and mix together for a total of 20 μL in a water bath. Heat the pot at 42°C for 15 minutes and 95°C for 3 minutes. When the synthesized cDNA needs to be stored for a long time, please store it at -20°C or lower temperature.

4. Real-Time PCR

法进行数据的相对定量分析：(1)用SuperReal PreMix Plus(SYBR Green)(货号：FP205)，进行扩增，实验操作按产品说明书进行。Real-Time反应体系如表10所示：Using an ABI 7300 fluorescence quantitative PCR instrument, the

The relative quantitative analysis of the data was carried out by the method: (1) SuperReal PreMix Plus (SYBR Green) (Item No.: FP205) was used for amplification, and the experimental operation was carried out according to the product instructions. The Real-Time reaction system is shown in Table 10:

Table 10 Real-Time PCR reaction system

reagent Usage amount 2 x SuperReal PreMix Plus 10μL Upstream primer (10μM) 0.6μL Downstream primer (10μM) 0.6μL 50×ROX Reference Dye^△ 2μL DNA template 2μL Sterilized distilled water 4.8μL

(2) The amplification program is: 95°C for 15min, (95°C for 10sec, 55°C for 30sec, 72°C for 32sec)×40 cycles, 95°C for 15sec, 60°C for 60sec, 95°C for 15sec).

(3) Primer screening

After mixing the cDNA of each sample, use this as a template for 10-fold gradient dilution. After dilution, take 2 μL of each sample as a template, and use the primers of the target gene and the internal reference gene for amplification respectively (see Table 11). Perform melting curve analysis, and screen primers according to the principle of high amplification efficiency and single-peak melting curve.

Table 11 Primer Screening Standard Curve Real-TimePCR Design

(4) Real-Time PCR detection of samples

After 20-fold dilution of the cDNA of each sample, 2 μL of the cDNA was taken as a template, and the primers of the target gene and the internal reference gene were used for amplification respectively (see Table 12). Meanwhile, dissolution curve analysis was performed at 60-95°C.

Table 12 Sample Real-Time PCR detection design

template sample cDNA sample cDNA Repeated detection of the number of channels 3 3 primer target gene primer Internal reference gene primer

5. Statistics

Arrange the original ct values of the original results exported by running the program in the order of loading to obtain the original ct values of three replicate wells of each gene in each sample, and obtain the three replicate wells of the target gene and the internal reference gene in excel respectively. The average value of ct values was used to calculate the expression of the target gene in the control group (paracancerous tissue) and the experimental group (gastric cancer tissue) relative to the internal reference gene.

2. Experimental results

The statistical results are shown in Figure 2. Compared with adjacent tissues, the expression of LINC01895 was down-regulated in gastric cancer tissue samples.

Example 4 Diagnostic efficacy verification of LINC01895

Receiver operating curve (ROC) was drawn by SPSS software, AUC value, sensitivity and specificity were analyzed, and the diagnostic efficacy of the indicators alone was judged.

As shown in Table 13 and Figure 3, LINC01895 has high diagnostic performance, suggesting that LINC01895 can be used to diagnose gastric cancer.

Table 13 LINC01895 diagnostic efficacy data

Gene AUC Sensitivity specificity LINC01895 0.767 0.783 0.696

The preferred embodiments of the present application have been described in detail above with reference to the accompanying drawings. However, the present application is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application. These simple modifications all belong to the protection scope of the present application.

In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. The combination method will not be specified otherwise.

In addition, the various embodiments of the present application can also be combined arbitrarily, as long as they do not violate the idea of the present application, they should also be regarded as the content disclosed in the present application.

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cgcaggtaat aggtggcagt 20

Claims (6)

1. The application of a reagent for detecting the expression level of LINC01895 in a sample in the preparation of a product for diagnosing gastric cancer or predicting the prognosis of gastric cancer patients using Notch1 inhibitor. 2 . The application according to claim 1 , wherein the reagent comprises a reagent for detecting the expression level of LINC01895 in the sample by sequencing technology, nucleic acid hybridization technology, and nucleic acid amplification technology. 3 . 3. The application according to claim 1 or 2, wherein the reagent comprises a primer for specific amplification of LINC01895, and the sequence of the primer for specific amplification of LINC01895 is as shown in SEQ ID NO.5～6 Show. 4. A product for diagnosing gastric cancer or predicting the prognosis of gastric cancer patients using a Notch1 inhibitor, characterized in that the product comprises a reagent for detecting the expression level of LINC01895 in a sample. 5. The product according to claim 4, wherein the product comprises a chip, a test kit, and a nucleic acid membrane strip. 6. The product of claim 4, further comprising reagents for processing samples.

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