CN111254197A

CN111254197A - Gastric adenocarcinoma molecular marker and application thereof

Info

Publication number: CN111254197A
Application number: CN202010109358.3A
Authority: CN
Inventors: 韩淑毅; 徐祎慧; 王军; 汪运山; 马晓丽; 郑燕; 郏雁飞; 王敏
Original assignee: JINAN CENTER HOSPITAL
Current assignee: JINAN CENTER HOSPITAL
Priority date: 2019-07-12
Filing date: 2020-02-22
Publication date: 2020-06-09
Anticipated expiration: 2040-02-22
Also published as: CN110229911A; CN111254197B

Abstract

The invention discloses a gastric cancer molecular marker and application thereof, wherein the gastric cancer molecular marker disclosed by the invention is RP11-366L20.2 or RP4-724E 16.2. The invention discloses application of a reagent for detecting molecular markers RP11-366L20.2 or RP4-724E16.2 in preparation of a product for diagnosing gastric adenocarcinoma. The invention also discloses a product for diagnosing gastric adenocarcinoma, which contains a reagent for detecting RP11-366L20.2 or RP4-724E16.2, and the invention also discloses application of RP11-366L20.2 or RP4-724E16.2 in preparing a medicament for treating gastric cancer.

Description

Gastric adenocarcinoma molecular marker and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to gastric adenocarcinoma molecular markers and application thereof, and particularly relates to gastric adenocarcinoma molecular markers RP11-366L20.2 and RP4-724E16.2 and application thereof.

Background

Gastric Cancer is a malignant tumor with the highest morbidity and mortality among digestive system tumors, and is globally the fourth most serious disease burden, and the third most serious Cancer related mortality among gastric Cancer related deaths (Siegel R L, Miller K D, Ahmedin Jemal DVM phd. Cancer statistics,2017[ J ]. CA Cancer J Clin,2017,67 (1)). For our country, stomach cancer is a prominent public health problem. The incidence of gastric cancer in men in China is ranked second after lung cancer, the incidence of gastric cancer in women in China is only lower than that of breast cancer and lung cancer, and third place, and the incidence of gastric cancer in the whole and the antegrade of the cause of death of cancer are the second highest malignant tumors in China (Chen W, Zheng R, Baade P D, et al. cancer stattics in China,2015[ J ]. CACACACANCER J Clin,2016,66: 115-type 132.). Although the surgical treatment mode of the gastric cancer is continuously improved, the current situation that most gastric cancer patients in China are already in the advanced stage when the patients are diagnosed, the five-year survival rate is obviously lower than that of early gastric cancer, the pathogenesis of the gastric cancer is deeply researched, and the search of a gastric cancer marker on the basis of the pathogenesis is an important way for improving the early diagnosis of the gastric cancer. In addition, although the number of available chemotherapeutic drugs is increasing and the technical means of immunotherapy, gene therapy and the like are continuously updated, the improvement effect of the treatment methods on the life cycle of patients is not significant overall, and the research on the molecular mechanism of the occurrence and development of gastric cancer needs to be strengthened and potential ideal diagnosis and treatment targets are screened from the research.

Long non-coding RNAs (1 ncRNAs) are long RNAs that do not code for proteins. 1ncRNAs are distinguished in length from the well known short-chain RNAs such as tRNAs, miRNAs, nucleolar RNA (snorRNAs), etc., and are typically greater than 200 nucleotides; unlike mRNAs that encode proteins, 1ncRNAs contain no Open Reading Frame (ORF). LncRNAs are widely present in eukaryotes, in larger numbers than protein-encoding genes, and thanks to deeper, more extensive, and more extensive RNA sequencing, more and more recent bioinformatics techniques, the discovery of 1ncRNAs is increasing (Clark M B, Mercer T R, Bussotti G, et al.quantitative gene profiling of long coding RNAs with targeted RNA sequencing [ J ]. Nat Methods,2015,12(4): 339.). Most 1ncRNAs are produced in the same way as mRNAs, are transcribed by RNA polymerase II, and usually have a 5 'cap structure and a 3' poly-A tail.

LncRNAs can be used as markers for different Cell states and different physiological and pathological stages (Wang K C, Chang H Y. molecular Mechanisms of Long nonacoding RNAs [ J ]. Mol Cell,2011, 43(6): 904-. 914.). A large number of researches reveal that the 1ncRNAs have differential expression in tumors, and the 1ncRNAs have great values in the aspects of tumor diagnosis, typing, prognosis evaluation, drug response and the like. At present, the specific role and master-slave relationship of lncRNA in the occurrence and development of gastric cancer are not clear, and research on lncRNA related to gastric adenocarcinoma is of great significance for diagnosis and treatment of gastric adenocarcinoma.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a molecular marker for early diagnosis of gastric adenocarcinoma, and whether a subject suffers from gastric adenocarcinoma can be judged by detecting the level of the molecular marker, so that a better way and a better method are found for diagnosis and targeted treatment of gastric adenocarcinoma.

"molecular marker" refers to a molecular indicator having a specific biological property, biochemical characteristic, or aspect, which can be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition.

In the present invention, "marker" refers to a parameter associated with one or more biological molecules (i.e., "molecular marker"), such as naturally or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA). "marker" in the context of the present invention also includes reference to a single parameter which may be calculated or otherwise obtained by taking into account expression data from two or more different markers.

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

the invention provides application of a reagent for detecting a molecular marker in preparation of a product for diagnosing gastric adenocarcinoma, wherein the molecular marker is RP11-366L20.2 and/or RP4-724E 16.2.

"RP 11-366L 20.2" is located on chromosome 12 with gene number ENSG00000197301, and includes RP11-366L20.2 gene and its homologues, mutations, and isoforms. The term encompasses full-length, unprocessed RP11-366L20.2, as well as any form of RP11-366L20.2 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RP11-366L 20.2. Currently, there are four transcripts from RP11-366L20.2, with transcript IDs ENST00000356215.2, ENST00000439236.2, ENST00000504038.2, and ENST00000536648.1, respectively. A representative sequence of RP11-366L20.2 is shown in ENST 00000356215.2.

"RP 4-724E 16.2" is located on chromosome 20 and has gene number ENSG00000197670, including RP4-724E16.2 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed RP4-724E16.2, as well as any form of RP4-724E16.2 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of RP4-724E 16.2. A representative sequence of RP4-724E16.2 is shown in ENST 00000424252.1.

Further, the reagent is a reagent that specifically binds to RP11-366L20.2 or RP4-724E 16.2.

Further, the reagent specifically binding to RP11-366L20.2 or RP4-724E16.2 is nucleic acid specifically binding to RP11-366L20.2 or RP4-724E 16.2.

Further, the nucleic acid that specifically binds to RP11-366L20.2 or RP4-724E16.2 is selected from the group consisting of:

primers for specifically amplifying RP11-366L20.2 or RP4-724E 16.2;

probes that specifically recognize RP11-366L20.2 or RP4-724E 16.2; or

Chips for specific analysis of RP11-366L20.2 or RP4-724E 16.2.

As used herein, "primer" means an oligonucleotide, whether naturally occurring or synthetically produced in a purified restriction digest, that serves as a synthesis origin when placed under conditions to induce synthesis of a primer extension product that is complementary to a nucleic acid strand, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer depends on many factors, including temperature, source of primer, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. Factors involved in determining the appropriate length of a primer will be readily known to those skilled in the art.

"Probe" refers to a molecule that selectively binds to a particular desired target biomolecule, such as a nucleotide transcript or protein encoded by or corresponding to an intrinsic gene. Probes may be synthesized by one skilled in the art, or may be derived from a suitable biological preparation. Probes can be specifically designed to label them. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. In a preferred embodiment, the molecules used as probes include RNA and DNA.

As the probe, a labeled probe in which a polynucleotide for cancer detection is labeled, such as a fluorescent label, a radioactive label, or a biotin label, can be used. Methods for labeling polynucleotides are known per se. The presence or absence of the test nucleic acid in the sample can be checked by: immobilizing the test nucleic acid or an amplification product thereof, hybridizing with the labeled probe, washing, and then measuring the label bound to the solid phase. Alternatively, the polynucleotide for cancer detection may be immobilized, a nucleic acid to be tested may be hybridized therewith, and the nucleic acid to be tested bound to the solid phase may be detected using a labeled probe or the like. In this case, the polynucleotide for cancer detection bound to the solid phase is also referred to as a probe. Methods for assaying test nucleic acids using polynucleotide probes are also well known in the art. The process can be carried out as follows: the polynucleotide probe is contacted with the test nucleic acid at or near Tm (preferably within ± 4 ℃) in a buffer for hybridization, washed, and the hybridized labeled probe or template nucleic acid bound to the solid phase probe is then measured.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the coding region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of a target gene.

The primers or probes of the invention can be chemically synthesized using a solid phase support of phosphoramidite or other well known methods. The nucleic acid sequence may also be modified using a number of means known in the art. Non-limiting examples of such modifications are methylation, capping, substitution with one or more analogs of a natural nucleotide, and modification between nucleotides, for example, modification of an uncharged linker (e.g., methyl phosphate, phosphotriester, phosphoimide, carbamate, etc.), or modification of a charged linker (e.g., phosphorothioate, phosphorodithioate, etc.).

Many expression detection methods use isolated RNA. The starting material is typically total RNA isolated from a biological sample, e.g. from a tumor or tumor cell line, respectively, and a corresponding normal tissue or cell line. If the source of the RNA is a primary tumor, RNA may be extracted from a frozen or preserved paraffin-embedded and fixed (e.g., formalin-fixed) tissue sample.

As used herein, "chip," also referred to as an "array," refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

The probe has a base sequence complementary to a specific base sequence of the 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.

Furthermore, the primer sequence of the specific amplification RP11-366L20.2 is shown as SEQ ID NO. 1-2, and the primer sequence of the specific amplification RP4-724E16.2 is shown as SEQ ID NO. 3-4.

The invention provides a product for diagnosing gastric adenocarcinoma, which comprises a reagent for detecting RP11-366L20.2 or RP4-724E 16.2.

Further, the product comprises a chip or a kit.

Further, the chip includes: a solid phase carrier and a probe which is attached to the solid phase carrier and specifically recognizes RP11-366L20.2 or RP4-724E 16.2. Specifically, suitable probes can be designed according to the lncRNA of the present invention, and are immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays as desired.

In the present invention, the solid phase carrier includes plastic products, microparticles, membrane carriers, and the like. The plastic products can be combined with antibodies or protein antigens through a non-covalent or physical adsorption mechanism, and the most common plastic products are small test tubes, small beads and micro reaction plates made of polystyrene; the micro-particles are microspheres or particles polymerized by high molecular monomers, the diameter of the micro-particles is more than micron, and the micro-particles are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and the combination capacity is large; the membrane carrier comprises microporous filter membranes such as a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane and the like.

Further, the kit comprises:

primers for specifically amplifying RP11-366L20.2 or RP4-724E 16.2;

probes that specifically recognize RP11-366L20.2 or RP4-724E 16.2; or

Chip for specific analysis of RP11-366L20.2 or RP4-724E16.2

Further, the kit also comprises a nucleic acid extraction reagent, a polymerase chain reaction reagent, a color developing agent or indicator, nucleic acid analysis software or an instruction for use.

Such a kit may employ, for example, a test strip, membrane, chip, tray, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The solid support of the kit can be, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, a particle, a precipitate, a gel, a polymer, a sheet, a sphere, a polysaccharide, a capillary, a film, a plate, or a slide.

The invention provides application of a molecular marker in preparing a pharmaceutical composition for treating gastric adenocarcinoma, wherein the molecular marker is RP11-366L20.2 and/or RP4-724E 16.2.

Further, the pharmaceutical composition comprises inhibitors of RP11-366L20.2 and/or RP4-724E 16.2. The inhibitor is selected from: an interference molecule which takes RP11-366L20.2 or RP4-724E16.2 as a target sequence and can inhibit the expression of RP11-366L20.2 or RP4-724E16.2 genes, and comprises: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is siRNA. In the specific embodiment of the invention, the sequence of the siRNA is shown in SEQ ID NO. 7-10.

The invention provides a pharmaceutical composition for treating gastric adenocarcinoma, which comprises siRNA inhibiting RP11-366L20.2 and/or RP4-724E 16.2.

Further, the sequence of the siRNA is shown in SEQ ID NO. 7-10.

Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier comprises (but is not limited to) diluents, binders, surfactants, humectants, adsorption carriers, lubricants, fillers, and disintegrating agents.

The pharmaceutical composition of the invention can also be used in combination with other drugs for the treatment of gastric adenocarcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

The invention provides application of RP11-366L20.2 or RP4-724E16.2 in constructing a prediction model of gastric adenocarcinoma.

The invention also provides application of RP11-366L20.2 or RP4-724E16.2 in screening potential drugs for treating gastric adenocarcinoma, and when the substance to be detected can obviously reduce the expression level of RP11-366L20.2 or RP4-724E16.2, the substance can be used as a candidate drug for treating gastric adenocarcinoma.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression level of RP11-366L20.2 or RP4-724E16.2 gene is related to gastric adenocarcinoma for the first time, and whether the subject suffers from gastric adenocarcinoma or not and the risk of suffering from gastric adenocarcinoma can be judged by detecting the expression level of RP11-366L20.2 or RP4-724E16.2 in a subject sample, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject, and meanwhile, the diagnosis is carried out by adopting the molecular marker, so that compared with the traditional diagnosis means, the diagnosis is more timely, more sensitive and more specific.

Drawings

FIG. 1 is a graph of the detection of molecular markers expressed in gastric adenocarcinoma tissue using QPCR; wherein Panel A is RP11-366L20.2 and Panel B is RP4-724E 16.2.

FIG. 2 is a graph showing the effect of RP11-366L20.2 or RP4-724E16.2 on gastric adenocarcinoma cells measured using a CCK-8 cell proliferation assay.

Detailed Description

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. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 QPCR detection of expression of RP11-366L20.2 or RP4-724E16.2 in gastric adenocarcinoma

1. 33 cases of gastric adenocarcinoma patients were collected and corresponding paracarcinoma tissue samples were collected, chemotherapy, radiotherapy and endocrine treatment were not performed before all patients, and expression levels of RP11-366L20.2 and RP4-724E16.2 were measured by QPCR.

2. RNA extraction

Total RNA extraction was performed using the Total RNA extraction kit for animal tissues (catalog No. DP431) from Tiangen.

1) Homogenizing treatment

Adding 300 mul of lysis solution RL into every 10-20mg of tissue, and thoroughly grinding the tissue by using a grinding pestle; subsequently, 590. mu.l RNase-Free ddH was added to the homogenate₂O and 10 mul of protease K, evenly mixing, and treating for 10-20 min at 56 ℃.

2) Centrifuging at 12,000rpm for 2-5min, and collecting supernatant.

3) Slowly adding 0.5 times of the volume of the supernatant of absolute ethyl alcohol, mixing, transferring the obtained solution and the precipitate into an adsorption column CR3 (the adsorption column is placed in a collecting pipe), centrifuging at 12,000rpm for 30s, discarding the waste liquid in the collecting pipe, and placing the adsorption column back into the collecting pipe.

4) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm for 30s, the waste liquid was discarded, and the adsorption column was returned to the collection tube.

5) 80. mu.l of DNase I working solution was added to the center of the adsorption column CR3, and the mixture was left at room temperature for 15 min.

6) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm for 30s, the waste liquid was discarded, and the adsorption column was returned to the collection tube.

7) The adsorption column CR3 was added with 500. mu.l of the rinsing solution RW, left to stand at room temperature for 2min, centrifuged at 12,000rpm for 30s, the waste solution was discarded, and the adsorption column CR3 was returned to the collection tube.

8) Repeat step 7).

9) Centrifuge at 12,000rpm for 2min and discard the waste. The adsorption column CR3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material.

10) Transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 30-100 μ l of RNase-Free ddH into the middle part of the adsorption membrane₂O, standing at room temperature for 2min, and centrifuging at 12,000rpm for 2min to obtain an RNA solution.

11) The integrity and purity of the RNA was examined.

3、QPCR

Primers were designed based on the gene sequences of RP11-366L20.2, RP4-724E16.2 and GADPH, and the primer sequences were as follows:

RP11-366L20.2：

a forward primer: 5'-AGTGTTCGTCCTGTTCAT-3' (SEQ ID NO.1)

Reverse primer: 5'-ATTGTTCTTCCTCTGTCATTAC-3' (SEQ ID NO.2)

RP4-724E16.2：

A forward primer: 5'-CTATGTGATAAAGGAAGATG-3' (SEQ ID NO.3)

Reverse primer: 5'-CTTGACCTGTATGTGTAA-3' (SEQ ID NO.4)

GAPDH：

A forward primer: 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO.5)

Reverse primer: 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO.6)

PCR was carried out using a Quant one-step reverse transcription-fluorescent quantitation kit (SYBR Green) from Tiangen (catalog No. NG105), and the reaction system and reaction conditions are shown in Table 1.

In the Thermal Cycler

PCR amplification is carried out on the Time System amplification instrument, after the reaction is finished, the amplification curve and the dissolution curve of Real Time PCR are confirmed, and relative quantification is carried out by the delta CT method.

TABLE 1 QPCR reaction System and reaction conditions

4. Statistical analysis

The experiment was repeated 3 times, and all data were expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using a two-sided Student's t test, and three and more groups were analyzed using one-way anova. All results were plotted using graphpad software, with P <0.05 defined as statistically significant differences. The variables RP11-366L20.2 and RP4-724E16.2 were analyzed by ROC using SPSS to determine the diagnostic potency, sensitivity and specificity of the genes.

5. Results

The QPCR results are shown in figure 1, compared with the tissues beside the cancer, the RP11-366L20.2 and the RP4-724E16.2 are both up-regulated in the gastric adenocarcinoma tissue, the RP11-366L20.2 is up-regulated by about 7.83 times, and the RP4-724E16.2 is up-regulated by about 2.93 times, the difference has statistical significance (P <0.05), and the result indicates that whether the subject has the gastric adenocarcinoma can be judged by detecting the level of the RP11-366L20.2 or the RP4-724E 16.2.

ROC analysis shows that RP11-366L20.2 or RP4-724E16.2 has a significant difference between gastric adenocarcinoma patients and normal controls, and the areas under the curves are 0.970 and 0.939 respectively, which indicates that the diagnosis of the gastric adenocarcinoma patients has higher specificity and sensitivity, and the specific analysis conditions are shown in the following table:

TABLE 1 area under the curve

a. Under the nonparametric assumption

b. Zero hypothesis: real area is 0.5

According to the relation between RP11-366L20.2 or RP4-724E16.2 and gastric adenocarcinoma, the interference RNA and shRNA targeting RP11-366L20.2 or RP4-724E16.2 can be designed to treat gastric adenocarcinoma.

Example 2 Effect of RP11-366L20.2 or RP4-724E16.2 on gastric adenocarcinoma cells

1. Cell culture

Human gastric cancer cell line HGC-27 in RPMI1640 medium containing 10% fetal bovine serum 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, passaged by conventional digestion with 0.25% EDTA-containing trypsin, and cells in logarithmic growth phase were taken for experiment.

2. Transfection

siRNNA against RP11-366L20.2 or RP4-724E16.2 was designed and synthesized by Shanghai Ji code pharmaceutical technology, Inc., and the control was general siRNA-NC.

The sequences of siRNA-RP11-366L2(SEQ ID NO. 7-8) and siRNA-RP4-724E16.2 (SEQ ID NO. 9-10) for silencing RP11-366L20.2 are shown below.

The sense strand is 5'-ACUGAAACAAUUACAAGACAC-3' (SEQ ID NO.7)

The antisense strand is 5'-GUCUUGUAAUUGUUUCAGUCC-3' (SEQ ID NO.8)

The sense strand is 5'-AACAGAUUCAGUUUUGCACAU-3' (SEQ ID NO.9)

The antisense strand is 5'-GUGCAAAACUGAAUCUGUUUC-3' (SEQ ID NO.10)

Lipofectamin from Invitrogen was used^TM2000, transfecting siRNA aiming at RP11-366L20.2 or RP4-724E16.2 to stomach cancer HGC-27 cells with logarithmic growth phase, preparing cells planted in a 6-well plate in an incubator in advance before cell transfection, changing the liquid of the cells in the 6-well plate and continuously culturing 24h after transfection, and the specific operation is carried out according to the instruction.

The experiment was divided into 3 groups, a blank control group (HGC-27), a negative control group (siRNA-NC) and an experimental group (transfected siRNA).

3. QPCR detection of expression levels of RP11-366L20.2 or RP4-724E16.2 in cells

Total RNA of cells is extracted by using TRNzol Universal total RNA extraction reagent of TIANGEN, and the specific steps are detailed in the instruction. The reverse transcription and real-time quantitative PCR detection steps of RNA were the same as in example 1.

4. MTS measures the influence of RP11-366L20.2 or RP4-724E16.2 on gastric cancer cell proliferation

After the cells in different groups are digested by pancreatin, the cells are resuspended and counted, and the cell concentration is adjusted to be l multiplied by 10⁵Perml, seeded at a density of 100. mu.L/well in 96-well plates, i.e.a cell count per well of 1X 10⁴Among them, gastric cancer cells transfected with siRNA-RP11-366L20.2 or RP4-724E16.2 served as experimental group, and cells transfected with siRNA-NC served as control group. After the cells are cultured for 48 hours, adding a CellTiter96AQ 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% CO₂And incubating for 4h in a cell culture box at 37 ℃, detecting by using a microplate reader, and recording the absorbance (A) value of 490nm wavelength.

5. Statistical analysis

All data are expressed as mean ± standard deviation (mean ± SD). Comparisons between two groups were performed using a two-sided Student's t test, and three and more groups were analyzed using one-way anova. All results were analyzed using GraphPad Software, with P <0.05 defined as statistically significant differences.

6. Results

The siRNA transfection results showed that, with the expression level of the blank control group RP11-366L20.2 as reference set to 1, the expression level of RP11-366L20.2 of the experiment group transfected with siRNA-RP11-366L20.2 (relative expression level of 0.170 ± 0.0458) was significantly reduced compared to the expression level of RP11-366L20.2 of the transfection blank control group (relative expression level of 1) and RP11-366L20.2 of the transfection siRNA-NC group (relative expression level of 0.953 ± 0.0351), and the difference had statistical significance (experiment group vs blank control group, P0.001; experiment group vs siRNA-NC group, P0.0014), while there was no significant difference between the siRNA-NC group and the blank control group (P0.148)

With the expression level of the blank control group RP4-724E16.2 as a reference set as 1, compared with the expression level of the RP4-724E16.2 of the transfected blank control group (relative expression level of 1) and the expression level of the RP4-724E16.2 of the transfected siRNA-NC group (relative expression level of 0.933 ± 0.0379), the expression level of the RP4-724E16.2 of the transfected RP4-724E16.2 of the experimental group (relative expression level of 0.357 ± 0.0833) was significantly reduced, and the difference was statistically significant (experimental group vs blank control group, P ═ 0.0055; experimental group vs siRNA-NC group, P ═ 0.0074), while there was no significant difference between the siRNA-NC group and the blank control group (P ═ 0.0928).

MTS cell proliferation activity results as shown in fig. 2, a 490: siRNA-RP11-366L20.2(0.864 + -0.0611); siRNA-RP4-724E16.2(0.93 +/-0.0628) is obviously lower than a control group (1.43 +/-0.0938) transfected with siRNA-NC, which indicates that RP11-366L20.2 or RP4-724E16.2 can influence the proliferation activity of gastric cancer cells, and indicates that the change of the expression level of RP11-366L20.2 or RP4-724E16.2 can change the proliferation capacity of gastric cancer cells, thereby providing a molecular strategy for the treatment of 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.

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<213> Artificial Sequence (Artificial Sequence)

<400>8

gucuuguaau uguuucaguc c 21

<210>9

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

aacagauuca guuuugcaca u 21

<210>10

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gugcaaaacu gaaucuguuu c 21

Claims

1. Application of a reagent for detecting a molecular marker in preparation of a product for diagnosing gastric adenocarcinoma is characterized in that the molecular marker is RP11-366L20.2 and/or RP4-724E 16.2.

2. The use of claim 1, wherein the agent is an agent that specifically binds RP11-366L20.2 or RP4-724E 16.2.

3. The use of claim 2, wherein the reagent that specifically binds RP11-366L20.2 or RP4-724E16.2 is a nucleic acid that specifically binds RP11-366L20.2 or RP4-724E 16.2;

preferably, the nucleic acid is selected from the group consisting of:

primers for specifically amplifying RP11-366L20.2 or RP4-724E 16.2;

probes that specifically recognize RP11-366L20.2 or RP4-724E 16.2; or

Chips for specific analysis of RP11-366L20.2 or RP4-724E 16.2.

4. The use of claim 3, wherein the primer sequence for specific amplification of RP11-366L20.2 is shown as SEQ ID No. 1-2, and the primer sequence for specific amplification of RP4-724E16.2 is shown as SEQ ID No. 3-4.

5. A product for diagnosing gastric adenocarcinoma, which comprises a reagent for detecting RP11-366L20.2 or RP4-724E16.2, and preferably comprises a chip or a kit.

6. The product of claim 5, wherein the chip comprises: a solid phase carrier and a probe which is attached to the solid phase carrier and specifically recognizes RP11-366L20.2 or RP4-724E 16.2.

7. The product of claim 5, wherein the kit comprises:

primers for specifically amplifying RP11-366L20.2 or RP4-724E 16.2;

probes that specifically recognize RP11-366L20.2 or RP4-724E 16.2; or

Chips for specific analysis of RP11-366L20.2 or RP4-724E 16.2;

preferably, the kit further comprises a nucleic acid extraction reagent, a polymerase chain reaction reagent, a color-developing agent or indicator, nucleic acid analysis software, or instructions for use.

8. The application of the molecular marker in preparing the pharmaceutical composition for treating gastric adenocarcinoma is characterized in that the molecular marker is RP11-366L20.2 and/or RP4-724E 16.2.

9. The use of claim 8, wherein the pharmaceutical composition comprises an inhibitor of RP11-366L20.2 and/or RP4-724E 16.2; preferably, the inhibitor is siRNA.

10. The pharmaceutical composition for treating gastric adenocarcinoma is characterized by comprising siRNA for inhibiting RP11-366L20.2 and/or RP4-724E16.2, and preferably, the sequence of the siRNA is shown as SEQ ID NO. 7-10.