CN106701986B - Application of the molecular marker in gastric cancer diagnosis and treatment - Google Patents

Abstract

The invention discloses application of the molecular marker in gastric cancer diagnosis and treatment, and specifically the lncRNA is ENSG00000228742, are in specificity overexpression in stomach organization.Experiments have shown that, the expression of ENSG00000228742 is reduced by specificity, the proliferation of stomach cancer cell can be effectively inhibited, prompt ENSG00000228742 can be used as the auxiliary diagnostic index of early gastric caacer diagnosis, and the expression of interference ENSG00000228742 gene can become the new target drone for the treatment of gastric cancer.

Description

Application of molecular markers in the diagnosis and treatment of gastric cancer

technical field

The invention belongs to the field of biomedicine, and relates to the application of molecular markers in the diagnosis and treatment of gastric cancer. The specific molecular marker is ENSG00000228742.

Background technique

Gastric cancer is one of the most common malignant tumors of the digestive tract. Although the incidence and mortality of gastric cancer have decreased in the world in recent years, it still ranks second and third among malignant tumors. The diagnosis and treatment of gastric cancer It is still a research hotspot of common concern for oncologists. China is a country with a high incidence of gastric cancer, and the incidence and mortality of gastric cancer in my country have long been among the highest in the world. Gastric cancer can be divided into early gastric cancer (EGC) and advanced gastric cancer (AGC). Early gastric cancer refers to gastric cancer limited to the mucosa and submucosa. The prognosis of early gastric cancer is good, the operation rate is lower than 5% to 10%, and the 5-year survival rate is over 90%, while the 5-year survival rate of patients with advanced gastric cancer is still less than 20%. Therefore, early diagnosis and early treatment of gastric cancer can improve the prognosis of patients. , play an important role in reducing mortality.

At present, the national cancer disease control strategy has shifted from passive treatment to active prevention and early identification. For gastric cancer, the focus is on the detection of precancerous lesions and the identification of high-risk groups, and at the same time, improving the detection of early gastric cancer and precancerous lesions by gastroscope detection rate. However, there is a big gap between the early diagnosis rate of gastric cancer in my country and advanced countries. The overall early diagnosis rate of gastric cancer is less than 10%. The vast majority of gastric cancer patients are in the advanced stage when they are diagnosed. The treatment costs are huge and the prognosis is poor. To come with a larger burden, the letter needs to be improved.

The difficulty of early diagnosis of gastric cancer is that most patients with early gastric cancer do not have specific clinical symptoms and signs, and may only present with non-specific epigastric discomfort, while commonly used tumor markers such as CEA and CA19-9 are sensitive and specific for the diagnosis of early gastric cancer. Sex is low. Therefore, finding new biological targets for the monitoring and intervention of gastric cancer has become an urgent problem to be solved in the prevention and treatment of gastric cancer.

LncRNAs are a class of endogenous RNA molecules with a fragment length of more than 200 nucleotides, lacking a complete and specific open reading frame and no ability to encode proteins. Most of them are transcribed by RNA polymerase II and generated by alternative splicing. LncRNAs are closely related to tumors, and abnormal expression and regulation of lncRNAs have been found in various tumors such as liver cancer, breast cancer, leukemia, and colon cancer. Various lncRNAs such as HEIR, HOTAIR, H19 and ANRIL have the function of proto-oncogene or promote tumor metastasis, while lncRNAs such as MEG3 and PTCSC3 have the function of tumor suppressor gene. The above facts indicate that lncRNA plays an important role in the occurrence and development of tumors. . Finding lncRNAs related to the occurrence and development of gastric cancer is of great significance for the prevention and treatment of gastric cancer and the study of its mechanism.

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 molecular marker that can be used for the diagnosis and treatment of gastric cancer. The molecular marker provided by the present invention is a lncRNA whose expression is up-regulated in gastric cancer patients and can be used as a Potential molecular targets for the diagnosis and treatment of gastric cancer.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention provides the use of ENSG00000228742 gene for preparing a pharmaceutical composition for preventing or treating gastric cancer.

Further, the pharmaceutical composition includes a downregulator of ENSG00000228742. Wherein, the down-regulating agent can down-regulate the expression of ENSG00000228742 gene at the gene level; the down-regulating agent of ENSG00000228742 is selected from: interfering molecules that take the ENSG0000018480 gene or its transcript as the target sequence and can inhibit the expression or gene transcription of the ENSG0000018480 gene , including: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, antisense nucleic acid, or a construct that can express or form said shRNA, small interfering RNA, microRNA, antisense nucleic acid .

Further, the down-regulating agent is selected from the siRNA of the following sequence: SEQ ID NO.8, SEQ ID NO.9.

The present invention provides a down-regulating agent of ENSG00000228742 for preventing or treating gastric cancer, and the down-regulating agent is siRNA.

The present invention provides a pharmaceutical composition for preventing or treating gastric cancer, the pharmaceutical composition contains:

A downregulator of ENSG00000228742 as described above; and

A pharmaceutically acceptable carrier.

The invention provides the use of ENSG00000228742 gene for screening potential substances for preventing or treating gastric cancer.

The present invention provides a method for screening potential substances for preventing or treating gastric cancer, the method comprising:

Treating a system expressing or containing the ENSG00000228742 gene with a candidate substance; and

Detect the expression of ENSG00000228742 gene in the system;

Wherein, if the candidate substance can reduce the expression or activity of ENSG00000228742 gene (preferably significantly reduced, such as lower than 20%, preferably lower than 50%, more preferably lower than 80%), it indicates that the candidate substance is a preventive measure or potential substances for the treatment of gastric cancer.

Further, the system includes (but is not limited to): cell system, subcellular system, solution system, tissue system, organ system or animal system.

The invention provides a use of ENSG00000228742 gene for preparing a product for diagnosing gastric cancer.

Further, the products include chips, preparations, or kits.

Further, the chip includes a probe that specifically recognizes ENSG00000228742; the kit includes a primer that specifically amplifies ENSG00000228742 or a probe or a chip that specifically recognizes ENSG00000228742.

Wherein, the chip can be used to detect the expression level of multiple genes including ENSG00000228742 gene (eg, multiple genes related to gastric cancer); the kit can be used to detect multiple genes including ENSG00000228742 gene (eg, multiple genes related to gastric cancer) , expression levels of multiple genes associated with gastric cancer.

Advantages and beneficial effects of the present invention:

The present invention discovers for the first time that the differential expression of ENSG00000228742 is related to the occurrence and development of gastric cancer. By detecting the expression of ENSG00000228742 in the sample of the subject, it can be judged whether the subject has gastric cancer, so as to guide the clinician to provide the subject with a preventive plan or treatment Program.

The present invention discovers a new molecular marker for gastric cancer-ENSG00000228742, and using the molecular marker to prevent or treat gastric cancer is more sensitive and specific.

Description of drawings

Fig. 1 is a graph showing the expression of ENSG00000228742 gene in gastric cancer tissue using QPCR;

Figure 2 is a graph of the expression of ENSG00000228742 in gastric cancer cells detected by QPCR;

Figure 3 is a graph showing the effect of ENSG00000228742 gene silencing on the expression level of this gene in gastric cancer cells using QPCR;

Figure 4 is a graph showing the effect of ENSG00000228742 gene on the proliferation of gastric cancer cells detected by MTS method.

specific implementation

After extensive and in-depth research, the present invention obtains lncRNA related to gastric cancer through lncRNA chip screening, and discovers for the first time that ENSG00000228742 exhibits specific high expression in gastric cancer. Experiments have shown that by specifically reducing the expression level of ENSG00000228742, it can effectively inhibit the proliferation of gastric cancer cells, suggesting that detecting the expression level of ENSG00000228742 gene can be one of the auxiliary diagnostic indicators for early diagnosis of gastric cancer. .

Molecular markers

The term "molecular marker" is any gene whose expression level in a tissue or cell is altered compared to that in a normal or healthy cell or tissue.

Those skilled in the art will recognize that the utility of the present invention is not limited to quantifying the gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, a marker gene may have the nucleotide sequence specified by SEQ ID NO.1. In some embodiments, it has a cDNA sequence that is at least 85% identical or similar to the listed sequence, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical or similar cDNA sequences.

The present invention can utilize any method known in the art to measure gene expression. As will be understood by those skilled in the art, the means of measuring gene expression is not an important aspect of the present invention. The expression levels of biomarkers can be detected at the transcriptional level.

In some embodiments, the expression level of the biomarker is detected at the transcriptional level. Various methods for specific DNA and RNA measurements using nucleic acid hybridization techniques are known to those of skill in the art. Some methods involve electrophoretic separation (eg, Southern blot for detection of DNA and Northern blot for detection of RNA), but DNA and RNA measurements (eg, by dot blot) can also be performed without electrophoretic separation. Southern blots of genomic DNA (eg, from humans) can be used to screen for restriction fragment length polymorphisms (RFLPs) to detect the presence of genetic disorders affecting the polypeptides of the invention. All forms of RNA can be detected.

pharmaceutical composition

Based on the findings of the present inventors, the present invention provides a use of ENSG00000228742 gene for preparing a pharmaceutical composition for preventing or treating gastric cancer. Wherein the pharmaceutical composition comprises an effective amount of a down-regulating agent of ENSG00000228742, and/or other drugs compatible with the accelerator, and a pharmaceutically acceptable carrier and/or adjuvant.

As used in the present invention, the down-regulators of ENSG00000228742 include (but are not limited to) inhibitors, blockers, nucleic acid inhibitors, etc., as long as they can down-regulate the expression level of the ENSG00000228742 gene at the gene level.

As a preferred mode of the present invention, the down-regulating agent of ENSG00000228742 is a small interfering RNA specific for ENSG00000228742. The "small interfering RNA" refers to a short-segment double-stranded RNA molecule, which can degrade specific mRNA by targeting mRNA with homologous complementary sequence. This process is the process of RNA interference (RNA interference). Small interfering RNAs can be prepared in the form of double-stranded nucleic acids, which contain a sense strand and an antisense strand, which only form double strands under hybridization conditions. A double-stranded RNA complex can be prepared from separate sense and antisense strands. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then hybridize by annealing, resulting in a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventors found the best effective fragment through a large number of alignment analysis. The inventors designed and synthesized a variety of siRNA sequences, and verified them by transfecting gastric cancer cell lines with transfection reagents respectively. As a result, two interference molecules with better interference effect were detected. They have SEQ ID NO. 8 and SEQ ID NO. The sequence shown in ID NO.9 was further tested at the cellular level, and the result proved that the inhibition efficiency was very high for the cellular experiment.

The nucleic acid inhibitors of the present invention, such as siRNA, can be chemically synthesized or prepared by transcribing into single-stranded RNA from an expression cassette in a recombinant nucleic acid construct. Nucleic acid inhibitors, such as siRNA, can be delivered into cells using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

As an optional mode of the present invention, the regulator of ENSG00000228742 can also be a "small hairpin RNA", which is a non-coding small RNA molecule capable of forming a hairpin structure, and the small hairpin RNA can pass through the RNA Interfering with pathways to suppress gene expression. As described above, shRNA can be expressed from a double-stranded DNA template encoding the desired transcript. The double-stranded DNA template encoding the desired transcript is inserted into a vector, such as a plasmid or viral vector, and then ligated to a promoter for expression in vitro or in vivo. Under the action of DICER enzyme in eukaryotic cells, shRNA can be cleaved into small interfering RNA molecules to enter the RNAi pathway. "shRNA expression vector" refers to some plasmids that are routinely used in the art to construct shRNA structures, usually there are "spacer sequences" and multiple cloning sites or replacement sequences on both sides of the "spacer sequence", so that people can use shRNA The corresponding DNA sequence (or analog) is inserted into the multi-cloning site by forward and reverse manners or the replacement sequence on it is replaced, and the RNA after the transcription of the DNA sequence can form a shRNA (Short Hairpin) structure. The "shRNA expression vector" can be purchased through commercial channels, for example, some viral vectors.

The gene expression vector can be various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The introduction of the expression vector into the host cell can use electroporation, calcium phosphate, liposome, DEAE dextran, microinjection, viral infection, lipofection, and binding to cell membrane-permeable peptides well-known methods.

Those skilled in the art can use well-known methods to construct the expression vector required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as karamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, the term "host cell" includes both prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells and mammalian cells. Preferably, the host cells are eukaryotic cells, such as CHO cells, COS cells and the like.

The term "effective amount" refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals. "Pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent and includes various excipients and diluents. The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The carrier may also contain cell transfection reagents.

In the present invention, various methods well known in the art can be used to administer the promoter or its transcribed gene, or its pharmaceutical composition to mammals. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained-release administration, etc.; preferably, the administration mode is parenteral administration.

Preferably, it can be carried out by means of gene therapy. For example, the down-regulating agent of ENSG00000228742 can be directly administered to the subject by methods such as injection; or, an expression unit (such as an expression vector or virus, or siRNA or shRNA) carrying the down-regulating agent of ENSG00000228742 can be delivered through a certain route To the target, the specific situation depends on the type of the down-regulator, which is well known to those skilled in the art.

The effective amount of the down-regulating agent of ENSG00000228742 according to the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the down-regulating agent of the ENSG00000228742 such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, route of administration, etc. For example, several divided doses may be administered daily, or the dose may be proportionally reduced, as dictated by the exigencies of the therapeutic situation.

drug screening

The invention provides the use of ENSG00000228742 in screening human gastric cancer diagnosis and treatment drugs. That is: treating a system expressing ENSG00000228742 with a candidate substance; and detecting the expression or activity of ENSG00000228742 in the system; if the candidate substance can down-regulate the expression or activity of ENSG00000228742, it indicates that the candidate substance is a potential substance to inhibit gastric cancer. The system expressing ENSG00000228742 can be, for example, a cell (or cell culture) system, and the cells can be cells that endogenously express ENSG00000228742; or can be cells that recombinantly express ENSG00000228742. The described system of expressing ENSG00000228742 can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, a rabbit, a sheep, a monkey, etc.)) Wait.

Detect the expression of the ENSG00000228742 gene in the system of the test group, and compare it with the control group, wherein the control group is a system that does not add the expression of the candidate substance or contains the ENSG00000228742 gene; if the expression of the ENSG00000228742 gene in the test group is statistically significant It is lower than the control group, indicating that the substance is a potential substance for the prevention or treatment of gastric cancer.

chip

The lncRNA chip of the present invention comprises: a solid phase carrier; and oligonucleotide probes fixed on the solid phase carrier in an orderly manner, the oligonucleotide probes specifically correspond to those shown in ENSG00000228742 part or all of the sequence.

Specifically, suitable probes can be designed according to the lncRNA of the present invention, and fixed on a solid phase carrier to form an "oligonucleotide array". The term "oligonucleotide array" refers to an array of addressable locations (ie locations characterized by distinct, accessible addresses), each addressable location having a characteristic associated therewith Oligonucleotides. The oligonucleotide array can be divided into multiple subarrays as desired.

The term "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 labeled, either directly or indirectly, to include primers. Hybridization means, including, but not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

The terms "complementary" or "complementarity" are used to refer to polynucleotides (ie, sequences of nucleotides) that are related by the principles of base pairing. For example, the sequence "5'-A-G-T-3'" is complementary to the sequence "3'-T-C-A-5'". Complementarity can be "partial," in which only some of the nucleic acid's bases match according to base pairing principles. Alternatively, there may also be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is especially important in amplification reactions and detection methods that rely on binding between nucleic acids.

The term "stringency" is used to refer to the conditions under which nucleic acid hybridization is carried out: temperature, ionic strength, and the presence of other compounds such as organic solvents. Under "low stringency conditions", a nucleic acid sequence of interest will be its exact complement, sequences with single base mismatches, closely related sequences (eg, sequences with 90% or greater homology) and only partial homology Sequences (eg, sequences with 50-90% homology) hybridize. Under "moderate stringency conditions", a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely related sequences (eg, 90% or greater homology). Under "high stringency conditions", a nucleic acid sequence of interest will hybridize only to its exact complement and (depending on conditions such as temperature) sequences with single base mismatches. In other words, under high stringency conditions, the temperature can be increased to exclude hybridization to sequences with single base mismatches.

The oligonucleotide probe for ENSG00000228742 gene in the present invention can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, and any length can be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The probes can be as short as 25, 20, 15, 13 or 10 bases in length. Likewise, the probes can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even entire genes. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs, which is consistent with the target nucleotide sequence. Complementary lengths are optimally 15-25 base pairs. The self-complementary sequence of the probe is preferably less than 4 base pairs, so as not to affect the hybridization efficiency.

The solid phase carrier in the present invention can be made of various materials commonly used in the field of gene chips, such as but not limited to nylon membranes, glass slides or silicon wafers modified with active groups (such as aldehyde groups, amino groups, etc.), unmodified glass slides, etc. sheets, plastic sheets, etc.

The preparation of the ENSG00000228742 chip can adopt the conventional manufacturing method of biochips known in the art. For example, if the solid phase carrier is a modified glass slide or silicon wafer, and the 5' end of the probe contains an amino-modified poly-dT string, the oligonucleotide probe can be prepared into a solution, and then spotted with a spotter The lncRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on the modified glass slide or silicon wafer, and then fixing by placing overnight.

Reagent test kit

The present invention provides a kit, which can be used to detect the expression of ENSG00000228742.

Preferably, the preparation or kit further contains a marker for labeling the RNA sample, and a substrate corresponding to the marker. In addition, the kit can also include various reagents required for RNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution , color developing solution, lotion, etc. In addition, the kit also includes instructions for use and/or chip image analysis software.

The medicaments in the present invention may also be administered alone with other therapeutic compounds in separate compositions or in dosage forms other than the main active ingredient. Partial doses of the principal ingredient may be administered concurrently with other therapeutic compounds, while other doses may be administered alone. During the course of treatment, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the present 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 term "sample" is used in its broadest sense. In one meaning, it is intended to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and encompass liquids, solids, tissues and gases. Biological samples include blood products such as plasma, serum, and the like. However, such samples should not be construed as limiting the types of samples suitable for use in the present invention.

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 method of unreceipted specific conditions in the embodiment, usually according to conventional conditions, such as people such as Sambrook, molecular cloning: the conditions described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1 Screening of gene markers related to gastric cancer

1. Sample collection

8 cases of gastric cancer adjacent tissue and gastric cancer tissue samples were collected respectively. All cases did not receive chemotherapy and radiotherapy before surgery. All patients gave informed consent and obtained the consent of the organizational ethics committee.

2. Preparation of RNA samples (using EZNA kit to operate)

1) Take out clinical specimens from -80℃ ultra-low temperature freezer and thaw them on ice. About 20 mg of tissue was weighed and placed in a 2 mL centrifuge tube, and 1 mL of Trizol was added on ice. The tissue was fully homogenized to no solids using a hand-held automatic homogenizer, and left to stand at room temperature for 10 min.

2) Centrifuge at 13000rpm and 4°C for 10min.

3) Carefully transfer the supernatant to a clean RNase-Free 1.5mL centrifuge tube with a pipette.

4) Add 0.2 mL of dichloromethane, shake with a vortex mixer for 15 s, and stand at room temperature for 10 min.

5) Centrifuge at 3000rpm and 4°C for 10min.

6) Carefully pipette the aqueous phase and transfer to a 1.5 mL RNase-free centrifuge tube.

7) Add the same volume of isopropanol as in step 6), shake with a vortex mixer for 4 s to precipitate total RNA, and stand at room temperature for 30 min.

8) Centrifuge at 13000rpm for 10min at 4°C and discard the supernatant. Add 1 mL of 75% alcohol and mix again. Centrifuge at 13000rpm for 10min at 4°C, discard the supernatant.

9) Dry at room temperature for 10 min, add 12 μL of RNase-Free water to dissolve the RNA and place on ice for later use.

3. Total RNA Quantification and Purity Analysis

Optical density values of total RNA at 280 nm and 260 nm were determined using a Bio-Red UV spectrophotometer. The amount of RNA is calculated as 1OD _260nm -40μg RNA. When the value of OD ₂₆₀ /OD ₂₈₀ is between 1.8 and 2.0, the purity of total RNA is considered to be reliable and can be used in the next experiment.

4. lncRNA expression chip analysis

The Arraystar Human 1ncRNA Array of Arraystar Company was used to detect the differences of lncRNA expression profiles in gastric cancer tissues and adjacent tissues. Arraystar 1ncRNA chip design probes are oligonucleotides with a length of 60 nt. These long oligonucleotide probes can obtain ideal experimental results with high sensitivity and specificity under highly stringent hybridization conditions. Multiple probes are designed for each sequence, increasing the reliability of the signal.

5. Data analysis

Agilent GeneSpring software was used to analyze the chip results, and the lncRNAs with significantly different expression levels (the standard was that the expression level of the lncRNA in the cancer and adjacent to the cancer was more than 2 times different, and p<0.05) were screened.

6. Results

The results showed that the expression level of ENSG00000228742 in gastric cancer tissues was significantly higher than that in adjacent tissues.

Example 2QPCR sequencing to verify the differential expression of ENSG00000228742 gene

1. Perform large-sample QPCR verification on the differential expression of ENSG00000228742 gene. According to the sample collection method in Example 1, 50 gastric cancer tissues and 50 gastric cancer tissues were selected.

2. The RNA extraction procedure was as described in Example 1.

3. Reverse transcription

1) Reaction system:

1 μl of RNA template, 1 μl of random primers, added to 12 μl of double-distilled water, mixed well, centrifuged at low speed, 65°C for 5 min, and then cooled on ice.

Continue adding the following components to the 12 μl reaction:

4μl of 5× reaction buffer, 1μl of RNase inhibitor (20U/μl), 2μl of 10mM dNTP mixture, 1μl of AMV reverse transcriptase (200U/μl); fully mixed and centrifuged;

2) Reverse transcription reaction conditions

25°C for 5 minutes, 42°C for 60 minutes, 70°C for 5 minutes, and 4°C for 60 minutes.

3) polymerase chain reaction

Primer Design:

QPCR amplification primers were designed according to the sequences of ENSG00000228742 gene and GAPDH gene in Genebank, and were synthesized by Shanghai Bioengineering Co., Ltd. The specific primer sequences are as follows:

ENSG00000228742 gene:

The forward primer is 5'-CTCTTCAGCACAGAATCAG-3' (SEQ ID NO.2);

The reverse primer was 5'-ACTACAACCTACTCAATTACATT-3' (SEQ ID NO. 3).

GAPDH gene:

The forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO.4);

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

Prepare PCR reaction system:

2×qPCR mixture 12.5μl, gene primer 2.0μl, reverse transcription product 2.5μl, ddH ₂ O 8.0μl.

PCR reaction conditions: 95°C for 10 min, (95°C for 15s, 60°C for 60s) × 40 cycles, and 60°C for 5min extension reaction. . Using SYBR Green as a fluorescent marker, the PCR reaction was performed on a Light Cycler fluorescence quantitative PCR instrument, and the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was performed by ΔΔCT method.

5. Statistical methods

The experiments were repeated three times, and the results were expressed as mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The paired comparison between cancer and adjacent tissue was performed using t test. Statistical significance was considered when P<0.05.

6. Results

The results are shown in Figure 1. Compared with adjacent gastric cancer tissues, ENSG00000228742 was up-regulated in gastric cancer tissues, and the difference was statistically significant (P<0.05), which was consistent with the microarray detection results.

Example 3 Expression of ENSG00000228742 in gastric cancer cells

1. Cell culture

Human immortalized gastric mucosa epithelial cell line GES-1, human gastric cancer cell line HGC-27, MGC-803, and AGS (all purchased from Guangzhou Ryder Biotechnology Co., Ltd.) were prepared in 10% fetal bovine serum and 1% P/S The RPMI1640 culture is based on an incubator at 37°C, 5% CO ₂ , and 90% relative humidity. The medium was changed once every 2-3 days, and the cells were routinely digested and passaged with 0.25% EDTA-containing trypsin, and the cells in the logarithmic growth phase were taken for experiments.

2. RNA extraction

Discard the complete medium, wash twice with PBS, digest the cells with trypsin, make a cell suspension and add it to a 2 mL centrifuge tube, centrifuge at 1200 rpm for 8 min, discard the supernatant, add 1 mL of Trizol, and pipet repeatedly for more than 15 times with a pipette. Fully lyse cells. The rest of the operation steps are the same as those in Example 1.

3. Reverse transcription

The specific steps are the same as in Example 2.

4. Statistical methods

The experiments were repeated three times, and the results were expressed in the form of mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The difference between the two was tested by t test. Statistical significance was established when P<0.05.

5. Results

The results are shown in Figure 2. Compared with gastric mucosal epithelial cells, the expression of ENSG00000228742 gene was up-regulated in gastric cancer cells HGC-27, MGC-803 and AGS, and the difference was statistically significant (P<0.05), the same as the RNA-sep results. Consistent.

Example 4 Silencing of the ENSG00000228742 gene

1. The cell culture steps were the same as those in Example 3.

2. Design of siRNA

Negative control siRNA sequence (siRNA-NC):

The sense strand is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.6)

The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.7)

siRNA-1:

The sense strand is 5'-AAAUGGUUUAUAGUACAAGAU-3' (SEQ ID NO. 8)

The antisense strand is 5'-CUUGUACUAUAAAACCAUUUGU-3' (SEQ ID NO.9)

siRNA-2:

The sense strand is 5'-UUUAGACUCCCAUUUGGAGAG-3' (SEQ ID NO. 10)

The antisense strand is 5'-CUCCAAAUGGGAGUCUAAAGG-3' (SEQ ID NO.11)

The cells were seeded into a six-well cell culture plate at 4×10 ⁴ /well, and the cells were cultured in a 37°C, 5% CO ₂ incubator for 24 h. Lipofectamine 2000 (purchased from Invitrogen) was used for transfection.

The experiment was divided into three groups: control group (HGC-27), negative control group (siRNA-NC) and experimental group (siRNA1, siRNA2). The negative control group siRNA had no homology to the sequence of ENSG00000228742 gene, and the concentration was 20nM /well, and transfections were performed separately at the same time.

3. QPCR detection of the transcription level of ENSG00000228742 gene

3.1 The extraction steps of total cell RNA are the same as those in Example 3.

3.2 The reverse transcription steps were the same as those in Example 2.

3.3 The steps of QPCR amplification were the same as those in Example 2.

4. Statistical methods

The experiments were repeated 3 times, and the results were expressed in the form of mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis. The difference between the ENSG00000228742 gene experimental group and the control group was calculated using t-test, considered statistically significant when P<0.05.

5. Results

The results are shown in Figure 3. Compared with the non-transfection group and the transfected siRNA-NC group, the experimental group could significantly reduce the expression of ENSG00000228742 gene, and the difference was statistically significant (P<0.05).

Example 5 The effect of ENSG00000228742 gene on the proliferation of gastric cancer cells

MTS assay was used to detect the effect of ENSG00000228742 gene on the proliferation of gastric cancer cells.

1. The steps of cell culture and transfection are the same as those of Example 4.

2. The treated cells in each group were resuspended and counted after trypsinization, and the cell concentration was adjusted to 1×10 ⁵ /ml and seeded in a 96-well plate at a density of 100 μL/well, that is, the number of cells per well was 1×10 ⁴ indivual.

3. After the cells reach the corresponding detection time points (0d, 24h, 48h, 72h, 96h), add CelITiter96AQ single-solution cell proliferation assay (MTS) reagent at 10 μL/well, and shake with a micro-shaker for 1-2 minutes; Incubate for 4h in a cell incubator at 37°C under %CO ₂ .

4. Read the plate with a microplate reader, and measure its absorbance (A) value at a wavelength of 490 nm.

5. Statistical analysis

Experiments were repeated three times, and SPSS18.0 statistical software was used for statistical analysis. The difference between the two was tested by t test, and it was considered statistically significant when P<0.05.

6. Results

The results are shown in Figure 4. Compared with the control group, the proliferation of cells in the experimental group was significantly inhibited after transfection with siRNA-1, and the difference was statistically significant (P<0.05), indicating that ENSG00000228742 has the effect of promoting cell proliferation.

The description of the above embodiment is only for understanding the method and the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

SEQUENCE LISTING

<110> Taishan Medical College

<120> Application of molecular markers in the diagnosis and treatment of gastric cancer

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 3049

<212> DNA

<213> Human Resources

<400> 1

gaaatttggg tgcagacagg cacacaggga gaacaccatg tcaacacgaa ggcagaagtt 60

ggagtgatga gtctacaagc caaggattgc cagcaattca tcagaggcca ggacagcagc 120

ctggggcagc cccctacatg gtaccaactc catcaacacc ttgatctcag acttccaacc 180

tccagaactg tccagccctc catgaatgag gaacttcctg gggccactag tgcatgctgc 240

agctcacaca gtcaactcca tccacacctc cacaaacagc tgccctttgg ccactactca 300

ggcgtaagga cacacccagc agggtatgcc ctctggaggt cagaccaggg aagaagccct 360

tgcacagtca ggagccagct cagcacctgt gatgctgaat tctatgtggc aacttgactg 420

gacgcaggtg ctcggattaa acctgatttc tgggtatgtc tgtgagggta tttccagagg 480

agattagaat ttgaatccgt ggattggcaa agttggtgtc tgccccagtg tgggtggtca 540

ccatctactc tcctgagggc ctgaagagaa caaaaggcaa aggaaggagg aattcgccct 600

taacttgctt gccgcctgcc tgcttgagca gagacatttc atcacatctt ctctggccct 660

cgactgagat tcacaccatt ggcttccctg gttctcaggc attttgactt ggactgaact 720

acaccattgg ctttcctggg ctccagcttg cacatagcag attgtgggat ttctcagcct 780

ctattattac aggcgccagt tcttcaaaat caacctctct ctctctgtct ctctctctct 840

ccctagatca cagtctggaa gtctgctcac agtctcaagg ttggcatatc ccctagagcc 900

cccagactcc tcatcccatg gagaccaaca tggccagggg agggccagag cagggccttc 960

taaatcacag accccagggc aggagcccct ccggtctgga tctaaggatg gtattttcaa 1020

caattcctaa atttttatcc taggctctcc aaatgggagt ctaaaggtct caaaacatgg 1080

tccagaagaa gtatgcaaaa atgtaaattc ctaggcccca acccagacct atgaatcaga 1140

tgatctgtat gggggttcaa ggaatctata ttattaaaca atcttcctaa atgacttgta 1200

ttcatactaa agcttgagaa ttgctatcaa acagctagaa gttggagtga agggtcctgc 1260

ctttcccatg aaattcatca actctatgcc ctcaaaagct gggctcttgg atgagctgct 1320

ctccattcta gagacattac cagctaggta gcctgctggt cctcaggaag ttaagcccag 1380

actcttcagc acagaatcag ataaacactg attacattgt actctacagt tctgagaaca 1440

ccattactta attcccggca agttgcaatg taattgagta ggttgtagtt ttttgggttt 1500

ggggtttttt tgtttttttaa atatatcagg cgtagttagc cagcttccta gaaacaccat 1560

gatctcttgg tgaaaattcc agaaatatgc atcacttcct aattaaagta gggaattttt 1620

aaaaattatt aaaccacatg ctttctaatt cctattgccc tttcttttca cccaccattc 1680

agcagtcagc agacactttc tgggtacaac aggtaggac catccaggca gaacaaaagc 1740

cattgatcat gaactacaga ggagagtttg gtaaatggac accagaaacc atggcctaca 1800

ggggcctggt tttctcccat ctaaatacta actaggccca acgctagttg ggcttagctt 1860

cctagatcag atgaaattgg gcacatttag ggttgggggt atggctctct attgtaagta 1920

gcaagagttt gagagaagaa aaaagggatt ttcttttttt tttcttttaa attttagagc 1980

attaacttta aacatcaaga tccaaacccc aaggattcag agaatcttgt actataaacc 2040

atttgtatta gtcagggttc tccagagaaa cagaacccat aggaggtata gagatagaga 2100

aatatggaat tggctcccat gattatggag gctgagaagt ccctcaatct gctgtctgtg 2160

agctggagac ctaggaaagc ttagctggtg gtacaaacca gcccaagtgc aaaggcccaa 2220

gaactaagag caccattgtc agagagcagg aaaagacaga tgtcccagct caaaaagaga 2280

gagcaaattc accttccttc caccatttta ttccgtcctg ttccattcct caacaggttg 2340

gatgatgagg gagatcttta tccatctcaa gtttttttgtg caaatatgga atacaaatga 2400

aaaatactta aagcacatca tcattgattt ccaacttaga ctagtttaac ttcccttttt 2460

cttctatcct gtcagttcct tgagggatcc tctctccttg ttcatctctg catctctagt 2520

aattagcaca gtgccaggca catggtaata tggctaaaac agtggattat tggtctccaa 2580

cataattaag cccatccaaa agaaaaacca acctcataaa tctgctttgc aaagttcata 2640

aacaatggat atgctatgtc tcaaaggtgc tggccaaaaa aatttaagaa cttgggaatt 2700

atgtgttatt ttgtttgttt ttttcaccaa gcctactttt ctgtcacaag agcccatatc 2760

aacaacacac ccatgtatgt ccttcttttt atctactttt ctccatgaag tttgtcacca 2820

tcaactgtta aaactgtaaa gatttgatca gctaaataag tggtctttga actaattcct 2880

ctcgtacttc ctaaaggaac tttgaaaaac tatgtatccc cttgcacatt ttcaagcaga 2940

cacctaagat ttttcatcat aaatttgaat aacttcaaaa tatgtactta ttagcatatt 3000

gtaaatattg atttttaaat aaaatatatc acactttgta tccaatgaa 3049

<210> 2

<211> 19

<212> DNA

<213> Artificial sequences

<400> 2

ctcttcagca cagaatcag 19

<210> 3

<211> 23

<212> DNA

<213> Artificial sequences

<400> 3

actacaacct actcaattac att 23

<210> 4

<211> 21

<212> DNA

<213> Artificial sequences

<400> 4

aatcccatca ccatcttcca g 21

<210> 5

<211> 19

<212> DNA

<213> Artificial sequences

<400> 5

gagccccagc cttctccat 19

<210> 6

<211> 19

<212> RNA

<213> Artificial sequences

<400> 6

uucuccgaac gugucacgu 19

<210> 7

<211> 19

<212> RNA

<213> Artificial sequences

<400> 7

acgugacacg uucggagaa 19

<210> 8

<211> 21

<212> RNA

<213> Artificial sequences

<400> 8

aaaugguuua uaguacaaga u 21

<210> 9

<211> 21

<212> RNA

<213> Artificial sequences

<400> 9

cuuguacuau aaaccauuug u 21

<210> 10

<211> 21

<212> RNA

<213> Artificial sequences

<400> 10

uuuagacucc cauuuggaga g 21

<210> 11

<211> 21

<212> RNA

<213> Artificial sequences

<400> 11

cuccaaaugg gagucuaaag g 21

Claims (3)

1. the purposes of the down-regulating agent of ENSG00000228742, is characterized in that, for preparing the pharmaceutical composition for the treatment of gastric cancer, described down-regulating agent is siRNA, wherein, the sense strand is SEQ ID NO.8, and the antisense strand is SEQ ID NO.8. 9. 2. A use of ENSG00000228742 gene, characterized in that it is used for screening potential substances for treating gastric cancer for non-therapeutic purposes. 3. A method for screening potential substances for the treatment of gastric cancer for non-therapeutic purposes, wherein the method comprises: Treating a system expressing or containing the ENSG00000228742 gene with a candidate substance; and Detect the expression of ENSG00000228742 gene in the system; Wherein, if the candidate substance can reduce the expression or activity of the ENSG00000228742 gene, it indicates that the candidate substance is a potential substance for the treatment of gastric cancer.