CN108251528B

CN108251528B - Application of LINC01814 in diagnosis and treatment of gastric cancer

Info

Publication number: CN108251528B
Application number: CN201810052867.XA
Authority: CN
Inventors: 李岚; 梁丽; 叶丽敏
Original assignee: Guizhou Provincial Peoples Hospital
Current assignee: Guizhou Provincial Peoples Hospital
Priority date: 2018-01-19
Filing date: 2018-01-19
Publication date: 2020-04-07
Anticipated expiration: 2038-01-19
Also published as: CN108251528A

Abstract

The invention discloses application of LINC01814 in diagnosis and treatment of gastric cancer. According to the invention, the QPCR experiment proves that the expression of LINC01814 is up-regulated in the gastric cancer tissue, and ROC analysis is carried out on the LINC01814, so that the diagnosis carried out by using the LINC01814 has higher accuracy. Cell experiments prove that the interference with the expression of LINC01814 can inhibit the proliferation of gastric cancer cells, and the LINC01814 can be used as a drug target to be applied to the personalized treatment of gastric cancer patients.

Description

Application of LINC01814 in diagnosis and treatment of gastric cancer

Technical Field

The invention belongs to the field of biological medicines, and relates to application of LINC01814 in diagnosis and treatment of gastric cancer.

Background

Gastric cancer is one of the most common malignancies, with tumor-related mortality rates third ranked worldwide. Although many gastric cancer patients can be treated properly and normatively and have better curative effect with the development of medical science, the improvement of operation modes and the improvement of medical diagnosis and treatment technologies, the early discovery of gastric cancer is difficult, and the treatment effect and the survival period of the gastric cancer patients are seriously influenced by the high recurrence rate and transfer rate after advanced gastric cancer resection. Compared with other tumors, the early-stage discovery rate of the gastric cancer is low, the advanced stage reaches more than 7% when diagnosis is confirmed, and the recurrence rate of patients in the advanced stage is higher after radical excision for 5 years. Therefore, while strengthening the early diagnosis research of the gastric cancer, the method explains the specific mechanism causing the occurrence and the metastasis of the gastric cancer, establishes a new diagnosis standard of the early gastric cancer or the metastatic gastric cancer, further explores new intervention measures, prevents the further relapse and the metastasis of the gastric cancer, improves the treatment effect before and after the operation of the gastric cancer, and increases the survival rate and the life quality of patients, thereby having great significance for the treatment and the prevention of the diseases.

The generation and development of gastric cancer are related to abnormal expression of oncogene and inactivation of cancer suppressor gene function, and gastric cancer generation and metastasis relate to multiple links such as cell dysfunction, virus-mediated immune microenvironment change, matrix destruction, angiogenesis and the like, including multi-factor induction and complex pathological process in which multiple genes participate. Therefore, searching for effective gene expression profiles and key protein markers related to the occurrence or metastasis of gastric cancer and predicting the occurrence of gastric cancer are important steps for ensuring the clinical treatment effect and improving the survival rate. With the development of high-throughput sequencing technology, researchers have demonstrated that RNA with protein-coding capacity only accounts for 5% of the human genome, but that most of the other 95% of non-coding regions are also transcribed, and that these transcripts are mostly long-and short-chain non-coding RNAs (ncrnas), and that most of the genome, which has long been considered as garbage, actually plays an important biological role. The abnormally expressed lncRNA can participate in various stages of tumor occurrence, development, invasion and metastasis through different pathways and different action mechanisms, and is a key factor of tumor progression.

The early symptoms of gastric cancer mostly lack specificity, the early cases account for only 10 to 20 percent of the surgical cases of gastric cancer, and approximately 2/3 patients are already in the middle and late stages or have lymph node and hematogenous metastasis at the time of diagnosis, and the 5-year survival rate is only 20 to 30 percent. Although the patents cn201710300189.x, CN201710069880.1 and CN201710069879.9 sequentially disclose that lncRNALINC01234, ENSG00000225521 and ENSG00000228742 can be applied to the diagnosis of gastric cancer, the clinical application still needs a long time, so that a new biological target is searched for the canceration monitoring and intervention of gastric cancer, and the method has important significance for the diagnosis and treatment of gastric cancer.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide the lncRNA biomarker for gastric cancer diagnosis and treatment.

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

the invention provides application of a reagent for detecting the LINC01814 level in preparation of a product for diagnosing gastric cancer.

Further, the product comprises a reagent for detecting the expression level of the LINC01814 gene in the sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology.

Further, the agent is selected from: a probe that specifically recognizes LINC 01814; or a primer that specifically amplifies LINC 01814.

Further, the sequence of the primer for specifically amplifying LINC01814 is shown in SEQ ID NO. 1-2.

The invention provides a product for diagnosing gastric cancer, which comprises a reagent for detecting LINC01814 level, and the product comprises a chip, a preparation or a kit.

Further, the agent is selected from:

a probe that specifically recognizes LINC 01814; or

Primers specifically amplifying LINC 01814.

The chip of the present invention can be used to detect the expression levels of a plurality of genes including the LINC01814 gene (e.g., a plurality of genes associated with gastric cancer); the kit of the present invention can be used to detect the expression levels of a plurality of genes including the LINC01814 gene (for example, a plurality of genes associated with gastric cancer).

The invention provides application of LINC01814 in preparation of a pharmaceutical composition for preventing or treating breast cancer.

Further, the pharmaceutical composition comprises a down-regulator of LINC 01814.

The down-regulating agent is selected from: an interfering molecule targeting LINC01814 or its transcript and capable of inhibiting LINC01814 gene expression or gene transcription, comprising: 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. Preferably, the down regulator is siRNA.

The invention provides a pharmaceutical composition, which comprises a downregulator of LINC01814, and/or other medicines compatible with the downregulator, and a pharmaceutically acceptable carrier and/or auxiliary material.

Further, the down regulator is siRNA.

Further, the siRNA has a sequence shown as SEQ ID NO. 7-8.

The medicaments of the invention can also be combined 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 invention has the advantages and beneficial effects that:

the invention discovers that the differential expression of LINC01814 is related to the occurrence and development of gastric cancer for the first time, and whether the subject has the gastric cancer can be judged by detecting the level of LINC01814 in a sample of the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC01814 gene in gastric cancer tissue by QPCR;

FIG. 2 is a graph showing the expression of LINC01814 in different gastric cancer cells by QPCR;

FIG. 3 is a graph showing the effect of an overexpression vector of LINC01814 gene on the expression level of the gene in gastric cancer cells by QPCR;

FIG. 4 is a graph showing the effect of LINC01814 gene on gastric cancer cell proliferation measured by MTS method.

Detailed Description

According to the invention, through extensive and intensive research, the expression levels of lncRNA in gastric cancer tissues and tissues beside the gastric cancer are detected by a high-throughput method, lncRNA with obvious expression difference is found, and the relation between the lncRNA and the occurrence and development of the gastric cancer is discussed, so that a better way and method are found for the early detection and the targeted treatment of the gastric cancer. Through screening, the invention discovers that LINC01814 is remarkably up-regulated in gastric cancer for the first time. Experiments prove that the siRNA interferes with LINC01814, can effectively inhibit the proliferation of gastric cancer cells, and provides a new way for personalized treatment of gastric cancer.

Biomarkers

The term "biomarker" is any gene whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the sequence of the marker gene is shown in LINC01814 gene (NR _110257.1) in GeneBank, the current International public nucleic acid database. LINC01814 in the present invention includes wild type, mutant or fragments thereof.

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

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 skilled in the art. Some methods involve electrophoretic separation (e.g., Southern blots for detecting DNA and Northern blots for detecting RNA), but measurements of DNA and RNA can also be made without electrophoretic separation (e.g., by dot blotting). Southern blots of genomic DNA (e.g., from humans) can be used to screen for Restriction Fragment Length Polymorphisms (RFLPs) to detect the presence of a genetic disorder affecting a polypeptide of the invention. All forms of RNA can be detected.

Chip and method for manufacturing the same

The lncRNA chip comprises: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in LINC 01814.

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.

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

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

The oligonucleotide probe of the invention directed against the LINC01814 gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. 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 at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

The solid phase carrier of the present invention can be made of various materials commonly used in the field of gene chip, such as but not limited to nylon membrane, glass or silicon slice modified by active group (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

The LINC01814 chip can be prepared by conventional methods for manufacturing biochips known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer using a spotting device, arranged into a predetermined sequence or array, and then fixed by standing overnight, so as to obtain the lncRNA chip of the present invention.

The invention provides a kit which can be used for detecting the expression of LINC 01814.

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

Down-regulating agent and pharmaceutical composition of LINC01814

Based on the inventors' findings, the present invention provides a functional expression down-regulator of LINC01814, the nature of which is not important to the present invention, as long as it inhibits the functional expression of LINC01814 gene, and which is useful as a substance for down-regulating LINC01814, for preventing or treating gastric cancer.

In the present invention, by "functional expression" of LINC01814 is meant the transcription and/or translation of a functional gene product. For non-protein encoding genes like LINC01814, "functional expression" can be deregulated at least two levels. First, at the DNA level, for example by deletion or disruption of the gene, or no transcription occurs (in both cases preventing synthesis of the relevant gene product). The loss of transcription can be caused, for example, by an epigenetic change (e.g., DNA methylation) or by a loss-of-function mutation. . As used herein, a "loss of function" or "LOF" mutation is a mutation that prevents, reduces or eliminates the function of a gene product relative to a gain-of-function mutation that confers enhanced or new activity to a protein. Functional deletions can be caused by a wide variety of mutation types, including but not limited to deletions of entire genes or gene portions, splice site mutations, frameshift mutations caused by small insertions and deletions, nonsense mutations, missense mutations replacing essential amino acids, and mutations that prevent proper cellular localization of the product. This definition also includes mutations in the promoter or regulatory region of the LINC01814 gene if these mutations interfere with the function of the gene. Null mutations are LOF mutations that completely disrupt the function of the gene product. Null mutations in one allele will typically reduce expression levels by 50% but may have a severe impact on the function of the gene product. It is noteworthy that functional expression may also be deregulated as a result of gain-of-function mutations: by conferring new activities to the protein, the normal function of the protein is deregulated and the expressed functionally active protein is reduced. Vice versa, functional expression may be increased, for example, by gene replication or by lack of DNA methylation. Functional expression can also be deregulated due to gain-of-function mutations: by conferring new activities to the protein, the normal function of the protein is deregulated and the expressed functionally active protein is reduced. Vice versa, functional expression may be increased, for example, by gene replication or by lack of DNA methylation.

Second, at the RNA level, for example by lack of efficient translation, for example because of instability of the mRNA (e.g. by UTR variants), can lead to degradation of the mRNA prior to translation of the transcript. Or by lack of efficient transcription, e.g. because mutations induce new splice variants.

As a preferred mode of the invention, the LINC01814 down-regulator is a LINC01814 specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by respectively transfecting a gastric cancer cell line with a transfection reagent, selects the siRNA with the best interference effect, and further performs experiments at a cell level, and the result proves that the siRNA can effectively inhibit the level of the LINC01814 gene in cells and the proliferation of gastric cancer cells.

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

Pharmaceutical composition

The pharmaceutical composition comprises a downregulator of LINC01814, and/or other medicines compatible with the downregulator, and a pharmaceutically acceptable carrier and/or auxiliary material.

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

In the present invention, the down-regulating agent, or a pharmaceutical composition thereof, can be administered to a mammal by various methods well known in the art. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

Preferably, it can be carried out by means of gene therapy. For example, a down-regulator of LINC01814 can be administered directly to a subject by a method such as injection; alternatively, the expression unit (e.g., expression vector or virus, etc.) carrying the downregulator of LINC01814 can be delivered to the target site by any route, depending on the type of downregulator in question, as is well known to those skilled in the art.

The effective amount of the LINC01814 down-regulator described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the LINC01814 down-regulator 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, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The term "sample" is used in its broadest sense. In one sense, specimens or cultures obtained from any source, as well as biological and environmental samples, are meant to be included. 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 type of sample that is suitable for use in the present invention.

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

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

Example 1 screening of Gene markers associated with gastric cancer

1. Sample collection

The collection of 8 paragastric cancer and gastric cancer tissue samples, respectively, did not receive chemotherapy or radiotherapy prior to surgery in all cases, all patients had known informed consent, and had informed consent from the tissue ethics committee.

2. Preparation of RNA samples

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

3. Total RNA quantitation and purity analysis

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

4. lncRNA expression chip analysis

The difference in lncRNA expression profiles between gastric cancer tissues and paracarcinoma tissues was examined using Arraystar Human 1ncRNA Array from Arraystar. The Arraystar 1ncRNA chip is designed with 60nt length oligonucleotide probe, and these long oligonucleotide probe can obtain high sensitivity and specificity ideal experiment result under high strict hybridization condition. A plurality of probes are designed for each sequence, so that the reliability of signals is improved.

5. Data analysis

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

6. Results

The results show that LINC01814 levels in gastric cancer tissues were significantly higher than in paracarcinoma tissues, with the difference being statistically significant (P < 0.05).

Example 2 QPCR sequencing validation of differential expression of LINC01814 Gene

1. Large sample QPCR validation was performed on the differential expression of LINC01814 gene. 50 cases of tissues adjacent to gastric cancer and gastric cancer were selected in the manner of sample collection in example 1.

2. RNA extraction

3. Reverse transcription

1) Reaction system:

1 mul of RNA template, 1 mul of random primer, and 12 mul of double distilled water are added, mixed evenly, centrifuged at low speed, cooled on ice at 65 ℃ for 5min, and the following components are added continuously: 5 × 4. mu.l of reaction buffer, 1. mu.l of RNase down-regulator (20U/. mu.l), 2. mu.l of 10mM dNTP mixture, and 1. mu.l of AMV reverse transcriptase (200U/. mu.l); fully and uniformly mixing and carrying out centrifugal treatment;

2) conditions for reverse transcription

5min at 25 ℃, 60min at 42 ℃, 5min at 70 ℃ and 60min at 4 ℃.

3) Polymerase chain reaction

Designing a primer:

QPCR amplification primers were designed based on the sequences of LINC01814 gene and GAPDH gene in Genebank and synthesized by Shanghai bioengineering, Inc. The specific primer sequences are as follows:

LINC01814 gene:

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

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

GAPDH gene:

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

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

Preparing a PCR reaction system:

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

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

4. ROC curve analysis

And analyzing the working characteristics of the testees of the LINC01814 by using a pROC package in the R language, calculating two accurate confidence spaces, and drawing an ROC curve.

5. Statistical method

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

6. Results

The results are shown in fig. 1, compared with the tissues adjacent to the gastric cancer, LINC01814 is up-regulated in the gastric cancer tissues, and the difference has statistical significance (P <0.05), which is consistent with the detection result of the chip; the ROC analysis result shows that the value of the area under the ROC curve (AUC) of LINC01814 is as high as 0.910125, and the LINC01814 serving as a biomarker is high in accuracy when being applied to clinical diagnosis of gastric cancer.

Example 3 expression of LINC01814 in gastric carcinoma cells

1. Cell culture

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

2. Extraction of RNA

Total RNA in cells was extracted using QIAGEN cell RNA extraction kit, the detailed steps are described in the specification.

3. Reverse transcription

The specific procedure is the same as in example 2.

4. Statistical method

The experiments were performed in 3 replicates, the results were represented as mean ± sd, and were statistically analyzed using SPSS18.0 statistical software, with the difference between the two using the t-test, and considered statistically significant when P < 0.05.

5. Results

As shown in FIG. 2, compared with gastric mucosal epithelial cells, the LINC01814 gene is up-regulated in gastric cancer cells HGC-27, MGC-803 and AGS, and the difference is statistically significant (P <0.05), wherein the difference in the HGC-27 cells is the most significant, so that the HGC-27 cells are selected for subsequent experiments to study the effect of LINC01814 on the gastric cancer cells.

Example 4 silencing of LINC01814 Gene

1. Cell culture procedure as in example 3

2. Design of siRNA

Designing siRNA aiming at the sequence of the LINC01814 gene, wherein the designed siRNA sequence is shown as follows:

negative control siRNA sequence (siRNA-NC):

sense strand: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.5),

antisense strand: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 6);

siRNA1：

sense strand: 5'-UUUGCUUUCGCGAAGUAGGAG-3' (SEQ ID NO.7),

antisense strand: 5'-CCUACUUCGCGAAAGCAAAAC-3' (SEQ ID NO. 8);

siRNA2：

sense strand: 5'-UUAGGAAUAAAUUCCAAUCUC-3' (SEQ ID NO.9),

antisense strand: 5'-GAUUGGAAUUUAUUCCUAAGC-3' (SEQ ID NO. 10);

siRNA3：

the sense strand is 5'-UUUGAACAAUCUAGAAGACAC-3' (SEQ ID NO.11),

the antisense strand is 5'-GUCUUCUAGAUUGUUCAAAGA-3' (SEQ ID NO.12)

siRNA4：

The sense strand is 5'-ACUCAUUGAAGACUUGUUCUA-3' (SEQ ID NO.13),

the antisense strand is 5'-GAACAAGUCUUCAAUGAGUGC-3' (SEQ ID NO.14)

3. Transfection

HGC-27 cells were packed at 2X 10⁵One well was inoculated into six well cell culture plates at 37 ℃ with 5% CO₂Culturing cells in an incubator for 24 h; transfection was performed in DMEM medium without double antibody containing 10% FBS according to the instructions of lipofectin 3000 (purchased from Invitrogen).

The experiment was divided into a blank control group (HGC-27), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2, siRNA3, siRNA4), wherein the siRNA of the negative control group had no homology with the sequence of LINC01814 gene at a concentration of 20 nM/well and was transfected separately.

4. QPCR detection of transcription level of LINC01814 gene

4.1 extraction of Total RNA from cells

4.2 reverse transcription procedure as in example 2.

4.3 QPCR amplification step as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, and the results were expressed as mean ± sd, and were statistically analyzed using SPSS18.0 statistical software.

6. Results

The results are shown in fig. 3, and compared with the HGC-27 and transfected unloaded siRNA-NC groups, the experimental group (siRNA 1-4) was able to reduce the level of LINC01814, wherein siRNA1 was able to significantly reduce the level of LINC01814, so siRNA1 was selected for subsequent experiments.

Example 5 Effect of LINC01814 Gene on gastric cancer cell proliferation

MTS experiment is adopted to detect the influence of LINC01814 gene on the proliferation capacity of gastric cancer cells.

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

2. Cells treated in each group were trypsinized, resuspended, counted, and adjusted to a cell concentration of lx 10⁵/ml

Inoculating to 96-well plate at a density of 100 μ L/well, i.e., 1X 10 cells per well⁴And (4) respectively.

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

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

5. Statistical analysis

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

6. Results

The results are shown in fig. 4, compared with the control group, the growth rate of the cells of the experimental group (siRNA1) is significantly lower than that of the cells of the control group, the difference is statistically significant (P <0.05), and the results indicate that the overexpression of LINC01814 promotes the proliferation of the gastric cancer cells.

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

Sequence listing

<110> Guizhou province people hospital

Application of <120> LINC01814 in diagnosis and treatment of gastric cancer

<160>14

<170>SIPOSequenceListing 1.0

<210>1

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

ccatatcctt gccaataca 19

<210>2

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ccaccatcac caataatca 19

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aatcccatca ccatcttcca g 21

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gagccccagc cttctccat 19

<210>6

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

uucuccgaac gugucacgu 19

<210>6

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

acgugacacg uucggagaa 19

<210>7

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

uuugcuuucg cgaaguagga g21

<210>8

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ccuacuucgc gaaagcaaaa c 21

<210>9

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

uuaggaauaa auuccaaucu c 21

<210>10

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gauuggaauu uauuccuaag c 21

<210>11

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

uuugaacaau cuagaagaca c 21

<210>12

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

gucuucuaga uuguucaaag a 21

<210>13

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

acucauugaa gacuuguucu a 21

<210>14

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gaacaagucu ucaaugagug c 21

Claims

1. Application of a reagent for detecting LINC01814 level in preparation of products for diagnosing gastric cancer.

2. The use according to claim 1, wherein the product comprises reagents for detecting the expression level of LINC01814 gene in the sample by sequencing, nucleic acid hybridization or nucleic acid amplification techniques.

3. Use according to claim 2, wherein said agent is selected from:

a probe that specifically recognizes LINC 01814; or

Primers specifically amplifying LINC 01814.

4. The use of claim 3, wherein the primer sequence for specific amplification of LINC01814 is shown as SEQ ID No. 1-2.

Use of a down-regulator of LINC01814 in the preparation of a pharmaceutical composition for the treatment of breast cancer.

6. The use of claim 5, wherein the down-regulating agent is an siRNA.

7. The use of claim 6, wherein the siRNA has a sequence as shown in SEQ ID No. 7-8.