CN107586850B

CN107586850B - Application of non-coding gene in diagnosis and treatment of liver cancer

Info

Publication number: CN107586850B
Application number: CN201711056992.XA
Authority: CN
Inventors: 宋宏涛; 马翠; 任静
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2020-06-23
Anticipated expiration: 2037-10-27
Also published as: CN107586850A

Abstract

The invention discloses application of a non-coding protein gene in diagnosis and treatment of liver cancer, and particularly relates to the non-coding protein gene LINC 01703. The invention discovers that LINC01703 is related to the occurrence and development of liver cancer for the first time. According to the invention, through bioinformatics analysis, the LINC01703 has a higher AUC value in a liver cancer patient, and the LINC01703 is prompted to have higher accuracy and specificity as a diagnosis marker of liver cancer. Experiments prove that the change of the expression level of LINC01703 can influence the proliferation, apoptosis and the like of liver cancer, and the LINC01703 can be used as a drug target for treating the liver cancer.

Description

Application of non-coding gene in diagnosis and treatment of liver cancer

Technical Field

The invention belongs to the field of biological medicines, relates to application of a non-coding protein gene in liver cancer diagnosis and treatment, and particularly relates to a non-coding protein gene LINC 01703.

Background

Hepatocellular carcinoma (HCC) is the most common malignancy worldwide, especially in asia, africa and southern europe. HCC has high malignancy and short survival time, and the 5-year survival rate is about 3 to 5 percent. The liver has abundant blood supply and lymphatic circulation, so that cancer cells are easy to spread and transfer, and the death rate of late-stage liver cancer is extremely high. New cases of the liver cancer in China account for about 45% of the world every year, annual death rates of malignant tumors account for the 1 st and 2 nd of the death rates of the malignant tumors in rural and urban areas respectively, and the liver cancer is found to be the country with the highest incidence rate. The occurrence of liver cancer seriously harms the health and life safety of people in China.

Surgery is still the most main method for treating liver cancer at present, however, HCC has hidden diseases, progresses rapidly, has no typical symptoms in early stage, and once HCC appears in middle and late stages, the opportunity of radical surgical treatment is usually lost, and the main problems of low surgical resection rate, high recurrence rate and high metastasis rate are also the main problems affecting the clinical efficacy of liver cancer. Although the treatment means mainly adopted clinically at present include the synergic radiotherapy and chemotherapy, the liver transplantation, the interventional therapy and the like except the surgical resection, the effect is not ideal. Furthermore, patients with advanced liver cancer often have basic diseases such as hepatic insufficiency and liver cirrhosis, which severely restrict the further application of cytotoxic chemotherapy drugs. The mechanism and biological process of hepatocellular carcinoma are very complex, and relate to the action of a plurality of factors such as external environment carcinogenesis, internal environment heredity and the like, including the activation of a cell molecular signal transduction pathway, the game of protooncogenes and cancer suppressor genes, the differentiation of tumor stem cells and the like. Therefore, the research of more effective new therapeutic means has become the focus of the current liver cancer treatment.

The molecular targeted medicine mainly interferes or blocks the efficacy of target molecules playing key roles in the processes of tumor occurrence, development and metastasis and related signal pathways thereof, thereby achieving the purpose of inhibiting tumor growth and metastasis. The molecular targeted therapy changes tumor characteristics into action targets, has the advantages of strong specificity, obvious curative effect, small adverse reaction and the like, and is the first choice of the current HCC treatment drugs. However, the action principle of the molecular targeted drugs is limited to the superficial curative effect, and the deep mechanism needs to be further researched. Therefore, the mechanism of the molecular targeted drug in treating cancer is deeply researched, a basis is provided for safer and more reasonable medication, a foundation is laid for further optimizing the drug, and the curative effect and the mechanism research of the molecular targeted drug enable the cure of HCC to be possible.

Disclosure of Invention

In order to make up for the defects of the prior art, one of the objects of the present invention is to provide a non-coding gene related to the occurrence and development of liver cancer, determine whether liver cancer or the risk of liver cancer is suffered by detecting the expression level of the gene, and realize the targeted therapy of liver cancer patients by changing the expression level of the gene.

The second objective of the present invention is to provide a method for screening potential drugs for treating liver cancer, which determines whether the candidate is a potential drug for treating liver cancer by adjusting the expression level of the biomarker.

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

the invention provides application of a reagent for detecting the expression level of LINC01703 in preparation of a product for diagnosing liver cancer, wherein the expression level of LINC01703 is up-regulated in a liver cancer patient.

Further, the agent is selected from:

a probe that specifically recognizes LINC 01703; or

And (3) specifically amplifying LINC01703 primers.

The invention provides a product for diagnosing liver cancer, which comprises a reagent for detecting the expression level of LINC01703 in a sample. The product provided by the invention is not limited to common detection products such as chips, nucleic acid membrane strips, preparations or kits, as long as the product can detect the expression level of LINC 01703. The "sample" includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, expectoration, alveolar bronchial lavage, urine, feces, etc. Preferably, the sample is tissue or blood.

Further, the reagent comprises a probe specifically recognizing LINC01703 or a primer specifically amplifying LINC 01703.

In a specific embodiment of the invention, the reagent comprises a primer for specifically amplifying LINC01703, and the sequence of the primer for specifically amplifying LINC01703 is shown as SEQ ID No. 2-3.

The invention provides application of LINC01703 gene in screening potential substances for preventing or treating liver cancer.

The invention provides a method for screening potential substances for preventing or treating liver cancer, which comprises the following steps:

treating a system expressing or containing the LINC01703 gene with a candidate substance; and

detecting the expression of the LINC01703 gene in the system;

wherein, if the candidate substance can reduce the expression level of the LINC01703 gene (preferably significantly reduced, such as more than 20%, preferably more than 50%, and more preferably more than 80%), the candidate substance is a potential substance for preventing or treating liver cancer. The system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

The candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed aiming at the LINC01703 gene or the upstream or downstream gene thereof.

The invention provides application of LINC01703 in preparation of a pharmaceutical composition for treating liver cancer.

Further, the pharmaceutical composition comprises an inhibitor of functional expression of LINC01703 selected from the group consisting of: an interfering molecule which uses LINC01703 or a transcript thereof as a target sequence and can inhibit LINC01703 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 inhibitor is siRNA.

Furthermore, the pharmaceutical composition also comprises other medicines compatible with the inhibitor and a pharmaceutically acceptable carrier and/or auxiliary material.

The invention provides a pharmaceutical composition for treating liver cancer, which comprises the following components:

an inhibitor of functional expression of LINC 01703; and

a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include, but are not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, salts, excipients, fillers, coagulants and conditioners, surfactants, dispersing agents, antifoaming agents.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC01703 in liver cancer patients by QPCR;

FIG. 2 is a graph of the differential expression of LINC01703 in liver cancer patients cross-validated using the TCGA database;

FIG. 3 is a ROC plot of LINC01703 in liver cancer patients;

FIG. 4 is a graph showing the detection of the expression of LINC01703 in hepatoma cells by QPCR;

FIG. 5 is a graph showing the effect of transfected siRNA on the expression of LINC01703 in hepatoma cells;

FIG. 6 is a graph of the effect of LINC01703 on cell proliferation measured using CCK 8;

FIG. 7 is a graph showing the effect of LINC01703 on apoptosis of hepatoma cells.

Detailed Description

The invention is widely and deeply researched, the expression level of lncRNA in liver cancer tissues and tissues beside the cancer is detected by adopting an lncRNA chip which covers the database most widely at present through a high-throughput method, lncRNA fragments with obvious expression difference are found, and the relation between the lncRNA fragments and the occurrence of liver cancer is discussed, so that a better way and a better method are found for the early detection and the targeted therapy of the liver cancer. Through screening, the invention discovers the significant up-regulation of LINC01703 in liver cancer for the first time. Experiments prove that the siRNA interferes with the silent LINC01703, can effectively inhibit the proliferation of liver cancer cells, and provides a new way for personalized treatment of liver cancer.

LINC01703 gene

The LINC01703 gene is located in the 1-band of the long arm 4 region of chromosome 1, and the nucleotide sequence of a representative human LINC01703 gene is shown as SEQ ID NO. 1. LINC01703 in the present invention includes wild type, mutant or fragments thereof. 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 target gene of the present invention.

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

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly known as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target nucleic acid sequence, transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of the target nucleic acid sequence under substantially constant conditions of temperature, ionic strength, and pH, where multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification, commonly known as NASBA, amplification of the probe molecule itself using RNA replicase (commonly known as Q β replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip, nucleic acid membrane strip and kit

The chip in the invention 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 01703.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

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

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, and may be polynucleotides obtained by replacing nucleotides in a part or all of them with artificial Nucleic acids such as PNA (polypeptide Nucleic Acid), LNA (registered trademark, locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (glyceronucleic Acid), and TNA (Threose Nucleic Acid).

In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

The invention provides a kit which can be used for detecting the expression level of LINC 01703. The reagent for detecting the expression level of LINC01703 in the specific embodiment of the invention comprises a primer for specifically amplifying LINC01703, and the sequence of the primer is shown as SEQ ID NO. 2-3. The kit also 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.

The gene detection kit or the gene chip can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to liver cancer) including the LINC01703 gene, and a plurality of markers of the liver cancer are simultaneously detected, so that the accuracy of liver cancer diagnosis can be greatly improved.

Inhibitors and pharmaceutical compositions

Based on the findings of the inventors, the present invention provides an inhibitor of functional expression of LINC01703, the nature of which is not important to the present invention, as long as it inhibits functional expression of LINC01703 gene, and these inhibitors are useful as substances for down-regulating LINC01703 and can be used for preventing or treating liver cancer.

As a preferred mode of the invention, the inhibitor of LINC01703 is a small interfering RNA molecule specific for LINC 01703. 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, verifies the siRNA sequences by transfecting a liver cancer cell line with a transfection reagent respectively, selects the siRNA with the best interference effect, and further performs experiments at a cell level, wherein results prove that the siRNA can effectively inhibit the expression level of LINC01703 gene in cells and the proliferation of liver 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.

The invention also provides a pharmaceutical composition which contains an effective amount of the LINC01703 inhibitor and a pharmaceutically acceptable carrier. The composition can be used for inhibiting liver cancer. Any of the foregoing inhibitors of LINC01703 may be used in the preparation of a pharmaceutical composition.

In the present invention, the pharmaceutically acceptable carriers include, but are not limited to, buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological salts, excipients, fillers, coagulants and conditioners, surfactants, dispersing agents, and antifoaming agents.

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administration of the 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 (host cell) transfection reagent.

The present invention may employ various methods well known in the art for administering the inhibitor or gene encoding the inhibitor, or pharmaceutical composition thereof, to a mammal. 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, an inhibitor of LINC01703 can be administered directly to a subject by a method such as injection; alternatively, an expression unit (e.g., an expression vector or virus, etc., or an siRNA or shRNA) carrying an inhibitor of LINC01703 can be delivered to a target site in a manner that allows expression of the active LINC01703 inhibitor, depending on the type of inhibitor, as will be appreciated by those skilled in the art.

The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In a specific embodiment, the pharmaceutical composition comprises at least one compound that inhibits the expression of LINC01703 gene and at least one chemotherapeutic agent. Chemotherapeutic agents for use in the present invention include, but are not limited to: microtubule activators, alkylating agents, antineoplastic antimetabolites, platinum-based compounds, DNA-alkylating agents, antineoplastic antibiotic agents, antimetabolites, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial and exotoxin agents.

Pharmaceutically acceptable carriers can include, but are not limited to: a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. Relevant delivery vehicles can include, but are not limited to: liposomes, biocompatible polymers (including natural and synthetic polymers), lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, inorganic (including metal) particles, and bacterial or viral (e.g., baculovirus, adenovirus, and retrovirus), phage, cosmid, or plasmid vectors.

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

The pharmaceutical compositions of the present invention may also be administered separately with other therapeutic compounds, either as separate compositions or in different dosage forms than the primary 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.

Statistical analysis

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

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 liver cancer

1. Sample collection

Cancer tissues and tissues adjacent to the cancer were collected from 10 patients with liver cancer, and the patients gave their informed consent, and all of the above specimens were obtained with the consent of 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. Reverse transcription and labelling

mRNA was reverse-transcribed into cDNA using the Low RNA Input Linear Amplification Kit, and the experimental group and the control group were labeled with Cy3, respectively.

4. Hybridization of

The gene chip adopts Kangcheng organism-Human lncRNA Array, and hybridization is carried out according to the steps of the chip use instruction.

5. Data processing

After hybridization, the chip was scanned with an Agilent scanner with a resolution of 5 μm, the scanner automatically scanned 1 time each with 100% and 10% PMT, and the results of 2 Agilent software were automatically merged. And (3) processing and analyzing the scanned image data by adopting Feature Extraction, and performing subsequent data processing on the obtained original data by applying a Bioconductor program package. Differential gene screening criteria: FDR<0.01，abs(log₂FC)>1.5。

6. Results

LINC01703 is up-regulated in liver cancer tissues compared to paracancerous tissues.

Example 2 QPCR sequencing validation of differential expression of LINC01703 Gene

1. Large sample QPCR validation was performed on the differential expression of LINC01703 gene. In example 1, 60 samples of liver cancer tissue and paracancerous tissue were collected.

2. The RNA extraction procedure was as in example 1.

3. Reverse transcription:

a25-mu-l reaction system is adopted, 1 mu g of total RNA is taken as template RNA for each sample, DEPC water, 5 × reverse transcription buffer solution, 10mM dNTP, 0.1mM DTT, 30 mu M Oligo dT, 200U/mu l M-MLV are respectively added into a PCR tube, the template RNA is incubated for 1h at the temperature of.42 ℃, is incubated for 10min at the temperature of 72 ℃ and is centrifuged for a short time.

(3) QPCR amplification assay

Designing amplification primer sequences of LINC01703 and housekeeping gene GAPDH, and synthesizing the primer sequences by Shanghai, wherein the primer sequences of the LINC01703 gene are shown as SEQ ID NO. 2-3; the primer sequence of housekeeping gene GAPDH is shown in SEQ ID NO. 4-5.

Prepare 25. mu.l reaction system: SYBR Green polymerase chain reaction system 12.5. mu.l, forward and reverse primers (5. mu.M) 1. mu.l each, template cDNA 2.0. mu.l, 8.5. mu.l enzyme-free water. All operations were performed on ice. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was carried out at 95 ℃ for 60s, (95 ℃ for 15s, 60 ℃ for 15s, 72 ℃ for 45s) for × 35 cycles.

SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent real-time 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.

3. Results

As shown in FIG. 1, the expression level of LINC01703 gene is up-regulated in liver cancer tissue compared with that of the tissue beside the cancer, and the difference is statistically significant (P <0.05), which is consistent with the result of RNA-sep.

Example 3 analysis of LINC01703 expression in TCGA database

1. Data collection

Collecting lncRNA expression profile data of 200 liver cancer tissues and 50 tissues beside the cancer from a TCGA database, and analyzing the expression level of LINC01703 in the liver cancer tissues and the tissues beside the cancer; box plots are drawn.

2. ROC curve analysis

And analyzing the working characteristics of the testees of the LINC01703 by using a pROC package in the R language, calculating a two-term accurate confidence space, and drawing an ROC curve.

3. Results

The expression level of LINC01703 is shown in fig. 2, and LINC01703 is significantly up-regulated in liver cancer tissues compared with the control group.

The ROC curve of LINC01703 is shown in figure 3, the AUC value of LINC01703 is as high as 0.905, and the LINC01703 has high specificity and sensitivity, which indicates that the LINC01703 has high accuracy when applied to diagnosis of liver cancer.

Example 4 differential expression of LINC01703 Gene in liver cancer cell lines

1. Cell culture

Human hepatoma cell lines HepG2, Huh7 and normal liver cell line HL-7702, cultured in DMEM medium containing 10% fetal calf 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 and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. Extraction of RNA

1) Digesting adherent cells by pancreatin, centrifuging, resuspending and cleaning the cells obtained by blowing, and then resuspending the cells in a DMEM culture medium containing 10% FBS;

2) transferring the resuspended cells to a 6-well plate, adding the culture medium to 2m 1/well, and slightly shaking the 6-well plate to uniformly resuspend the cells;

3) cells grow for 48 hours in an adherent manner, and the culture medium is removed;

4) cracking cells by using 1ml of Trizol reagent, repeatedly blowing and punching 6-hole plate walls, and completely cracking the cells as much as possible;

5) transfer cell lysates to 1.5ml DEPC treated EP tubes, and place on ice. 0.2m of 1 g of chloroform was added, and the remaining procedure was the same as that of the extraction of RNA from blood.

3. Reverse transcription

The specific procedure is the same as in example 2.

4. Results

As shown in FIG. 4, compared with the normal liver cell line, the LINC01703 gene is up-regulated in liver cancer cells HepG2 and Huh7, and the difference is statistically significant (P <0.05), which is consistent with the result of RNA-sep.

Example 5 silencing of LINC01703 Gene

1. Cell culture

Human hepatoma cell line HepG2 in DMEM medium containing 10% fetal calf 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 and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. SiRNA design

Designing an siRNA sequence aiming at the LINC01703 gene, wherein a negative control siRNA sequence (siRNA-NC) is shown as SEQ ID NO. 6-7; the sequence of the siRNA1 is shown in SEQ ID NO. 8-9; the sequence of the siRNA2 is shown in SEQ ID NO. 10-11; the sequence of the siRNA3 is shown in SEQ ID NO. 12-13.

Pressing the cells according to 2 × 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 2000 (purchased from Invitrogen).

The experiment was divided into a blank control group (HepG2), a negative control group (siRNA-NC) and an experimental group (20nM) (siRNA1, siRNA2, siRNA3), wherein the siRNA of the negative control group had no homology with the sequence of LINC01703 gene and had a concentration of 20 nM/well, and transfection was performed separately.

3. QPCR (quantitative polymerase chain reaction) detection of expression level of LINC01703 gene

3.1 extraction of Total RNA from cells

The specific procedure is the same as in example 4.

3.2 reverse transcription procedure as in example 2.

3.3QPCR amplification step as in example 2.

4. Results

The results are shown in fig. 5, compared with HepG2, transfection empty siRNA-NC, siRNA2 and siRNA3 groups, the siRNA1 group can significantly reduce the expression of LINC01703, and the difference has statistical significance (P < 0.05).

Example 6 CCK8 assay for cell proliferation

1. Cell culture and transfection procedures were as in example 4

2. CCK8 detection of cell proliferation

1) Grafting HepG2 cells in logarithmic proliferation phasePlanted in a 96-well plate, each hole is 2 × 10³(ii) individual cells;

2) the experiment is divided into three groups, namely a blank control group, a transfection siRNA-NC group and a transfection siRNA1, wherein each group is provided with 6 multiple holes;

3) adding 10 mul/well CCK8 reagent after transfection for 0h, 24h, 48h and 72h respectively;

4) after 2h, the absorbance of A450 was measured using a microplate reader.

3. Results

The results are shown in FIG. 6: the blank control group and the unloaded group have no obvious difference, the cell growth rate of the transfected siRNA1 group is obviously lower than that of the control group, the difference has statistical significance (P <0.05), and the results show that the expression of LINC01703 can promote the growth of the liver cancer cells.

Example 7 Effect of LINC01703 Gene on apoptosis of hepatoma cells

The effect of the LINC01703 gene on apoptosis was examined using flow cytometry.

1. The cell culture procedure was the same as in example 4.

2. The cell transfection procedure was as in example 5.

3. Step (ii) of

1) The 3m 110 × loading buffer was diluted with 27ml of distilled water.

2) Cell samples were collected and washed with pre-cooled PBS.

3) Cells were added to lml 1 × loading buffer, centrifuged at 300g for 10min and buffer aspirated.

4) The lml 1 × loading buffer was added again to adjust the cell concentration in the cell suspension to 1 × 10⁶One per ml.

5) The cell suspension was removed 100. mu.1 and added to the EP tube.

6) Add 5. mu.l Annexin V FITC to the EP tube, mix the liquid in the EP tube, incubate for 10min at room temperature in the dark.

7) Add 5. mu.1 PI stain to the EP tube and protect from light for 5min at room temperature.

8) Add 500. mu.l PBS solution to EP tube, mix gently, and detect by up-flow cytometry within 1 h. 4. As a result:

the results are shown in fig. 7, and the apoptosis rate of the experimental group is increased (P <0.05) compared with the control group, and the results show that the expression of LINC01703 inhibits the apoptosis of the liver 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> Beijing, the deep biometric information technology GmbH

Application of <120> non-coding gene in diagnosis and treatment of liver cancer

<160>13

<170>SIPOSequenceListing 1.0

<210>1

<211>882

<212>DNA

<213>Homo sapiens

<400>1

acgcccaccg ggtccgcccc cacgcggccg cctgcacagg gggttccagc cacccgcgct 60

ctcccaggtt tgccgacctt acgggccctt ctccgccagc cctggctggg tcacccccac 120

gacccgggac tcccccgccg ttccctcccg ggctgcctgc agtgaggtcc gctcctgagg 180

cctcagcctc gccgggggta gggccctctc ccgccgtcgc cgcagctcct ttccgtcccg 240

gggcctgggg ggcaccgtcc cacgggtgag ggccgcctcc agcgcctggc ccagccatgc 300

gaacgcctgg agcccgcgag ctgcagctgc ggccctgaag gtgccgcata tcctgccgag 360

tgggtgaagc gtggcacttt ggaaaacaaa tcgggtgatc cgcccacctc ggcctcccaa 420

agtgctggaa ttacaggcat gaaccacggt gcccggctga cacaaggaac tttctatgag 480

gtcctaggaa tgaaggaaaa caggcaataa ggcaagaatc gctgtcgaag gagtctgcgc 540

cacctgctcg cacagctcac ttgtgccttc tgagcctgct ggagcccagt gtacatatgc 600

cgtctccgcg tgattagaga ttgctgctgc acgtgcccaa tcccgtggcg gggctggaac 660

acaaacgggg tggtgttcca gctgcaacca tgcagatgaa gactataccc taggagatgg 720

aggaacaaca gacaagaaag aaacctgggt tcctgaataa ccacgtggag ccaagcagct 780

tacttatctc aagccactgt tgcccacctc cagattgaac tgtctgcaaa caaatcttta 840

cttgatgaga agtttaaata taattaaatc tagaatatta ca 882

<210>2

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

taggagatgg aggaacaa 18

<210>3

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aacagtggct tgagataa 18

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ccgggaaact gtggcgtgat gg 22

<210>5

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aggtggagga gtgggtgtcg ctgtt 25

<210>6

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

uucuccgaac gugucacgu 19

<210>7

<211>19

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

acgugacacg uucggagaa 19

<210>8

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

uguaauucca gcacuuuggg a 21

<210>9

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ccaaagugcu ggaauuacag g 21

<210>10

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

uguuuuccuu cauuccuagg a 21

<210>11

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

cuaggaauga aggaaaacag g 21

<210>12

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ucuuucuugu cuguuguucc u21

<210>13

<211>21

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gaacaacaga caagaaagaa a 21

Claims

1. Application of a reagent for quantitatively detecting LINC01703 expression level in liver tissues in preparation of products for diagnosing liver cancer.

2. The use according to claim 1, wherein the agent is selected from the group consisting of:

a probe that specifically recognizes LINC 01703; or

And (3) specifically amplifying LINC01703 primers.

3. The reagent for quantitatively detecting the LINC01703 gene expression level is applied to in-vitro screening of potential substances for preventing or treating liver cancer.

4. An in vitro screening method for potential substances for preventing or treating liver cancer, which comprises the following steps:

detecting the expression of the LINC01703 gene in the system;

wherein, if the candidate substance can reduce the expression level of the LINC01703 gene, the candidate substance is a potential substance for preventing or treating the liver cancer.

Application of an inhibitor of LINC01703 functional expression in preparing a pharmaceutical composition for treating liver cancer, wherein the inhibitor is interfering RNA.

6. The use of claim 5, wherein the pharmaceutical composition further comprises other drugs compatible with the inhibitor and a pharmaceutically acceptable carrier and/or adjuvant.