CN111440867B

CN111440867B - Application of biomarker in diagnosis and treatment of liver cancer

Info

Publication number: CN111440867B
Application number: CN202010149555.8A
Authority: CN
Inventors: 徐晓云; 陈圣雄; 姚冬梅
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2020-12-22
Anticipated expiration: 2040-03-06
Also published as: CN111440867A

Abstract

The invention discloses application of biomarkers in diagnosis and treatment of liver cancer. The invention discloses that the expressions of RP11-704M14.1 and RP11-498P14.5 are up-regulated in liver cancer, the expressions of HID1-AS1 are down-regulated in liver cancer, and further large sample QPCR and diagnosis efficiency verification are carried out, so that the application of the protein in diagnosis of liver cancer is found to have higher sensitivity and specificity, and especially the RP11-704M14.1 and RP11-498P14.5 are combined to generate synergistic diagnosis efficiency. The invention simultaneously proves that RP11-704M14.1, RP11-498P14.5 and HID1-AS1 play important roles in the occurrence and development of liver cancer through cell experiments, and can be used AS targets for treating the liver cancer.

Description

Application of biomarker in diagnosis and treatment of liver cancer

Technical Field

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

Background

Hepatocellular carcinoma (HCC) ranks third in all malignancies worldwide, and five-year survival rates of hepatoma patients remain low. Although diagnostic and therapeutic approaches for hepatocellular carcinoma have been continuously improved in recent years, the molecular mechanisms associated with the intrinsic tumor proliferation and invasion of hepatocellular carcinoma are complex, and more than sixty thousand patients die of hepatocellular carcinoma every year (Ferlay J, Shin H R, Bray F, et al. Because most liver Cancer cells have poor curative effect, high infection rate of chronic hepatitis B and long-term metastasis in clinical diagnosis, the liver Cancer is the most common malignant tumor below 60 years old in men (Chen W, Zheng R, Baade P D, et al. Cancer statistics in China,2015[ J ]. Ca Cancer J Clin,2016,66(2): 115-132.). Therefore, the molecular mechanism of liver cancer is deeply explored, and the development of scientific research on early diagnosis of hepatocellular carcinoma and early treatment of patients diagnosed with liver cancer is very important.

In recent years, with the adoption of high throughput testing of genetic engineering projects and human genome engineering project (human genome project HGP), it was found that less than 2% of genome is transcribed into RNA with coding function, and the rest is all non-coding RNA, wherein the total number of nucleotides of the RNA is more than 200, LncRNA, the sequence of LncRNA contains targets for mutual binding with other gene molecules, and the interaction further exerts relevant biological effects (St. laurent G, Wahlestedt C, Kapranov P. the Landscape of cloning RNA cloning [ J ] Trends in Genetics,2015,31(5): 239-. At present, more and more researches report that the lncRNA has complex biological functions, can regulate and control gene expression at multiple levels of epigenetics, transcription, post-transcription and the like, and is also closely related to the development of diseases. Recent studies have shown that LncRNA, a tumor Molecular marker for early diagnosis and treatment of tumors and prognosis judgment, can participate in the development and progression of malignant tumors by promoting or inhibiting the expression of other tumor-associated genes (Eulalio A, Mano M, Dal F M, et al. functional screening assays miRNAs indicating cardiac regeneration. [ J ]. Nature,2012,492(7429): 376. 381; Mattick J S, Rinn J L.discovery and identification of cloning NONCoding RNAs [ J ]. Nature Structural & Molecular Biology,2015,22(1): 5-7.).

At present, molecular markers have become the standard in the diagnosis of breast cancer and colorectal cancer, and are important steps in the treatment process. However, there is currently no uniform hepatocellular carcinoma biomarker standard. The research on the application of lncRNA related to the occurrence and development of hepatocellular carcinoma to the diagnosis and treatment of liver cancer is of great significance.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a biomarker for liver cancer diagnosis and treatment, whether a subject has liver cancer can be judged by detecting the expression level of the biomarker, and the biomarker can be used as a molecular target to be applied to personalized treatment of liver cancer.

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

the invention provides application of a reagent for detecting biomarkers in preparation of products for diagnosing hepatocellular carcinoma, wherein the biomarkers are selected from one or more of RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

Further, the biomarkers are RP11-704M14.1 and RP11-498P 14.5.

Furthermore, the product comprises a reagent for detecting the expression levels of the genes RP11-704M14.1, RP11-498P14.5 and HID1-AS1 in a sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology.

Further, the agent is selected from:

probes specifically recognizing RP11-704M14.1, RP11-498P14.5 and HID1-AS 1;

or primers for specifically amplifying RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

Further, primer sequences of specific amplification RP11-704M14.1, RP11-498P14.5 and HID1-AS1 are respectively shown AS SEQ ID NO. 1-2, SEQ ID NO. 3-4 and SEQ ID NO. 5-6.

The invention provides a liver cancer diagnosis product for in vitro detection of biological expression level, which comprises a chip, a kit and a nucleic acid membrane strip, wherein the biomarker is selected from one or more of RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

Further, the chip comprises oligonucleotide probes which specifically recognize RP11-704M14.1, RP11-498P14.5 and HID1-AS 1; the kit comprises primers for specifically amplifying RP11-704M14.1, RP11-498P14.5 and HID1-AS1 or oligonucleotide probes for specifically recognizing RP11-704M14.1, RP11-498P14.5 and HID1-AS 1; the nucleic acid membrane strip comprises oligonucleotide probes which specifically recognize RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

The invention provides application of biomarkers in constructing a calculation model for predicting hepatocellular carcinoma, wherein the biomarkers are selected from one or more of RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

The invention provides application of a biomarker in preparing a pharmaceutical composition for treating hepatocellular carcinoma, wherein the biomarker is selected from one or more of RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

Further, the pharmaceutical composition comprises inhibitors of RP11-704M14.1 and/or RP11-498P 14.5; or an enhancer of HID1-AS1, wherein the inhibitor can inhibit the expression level of RP11-704M14.1 or RP11-498P 14.5; the promoter can increase the expression level of HID1-AS 1.

Further, the inhibitor is siRNA which specifically inhibits the biomarker.

Further, the promoter is a vector for specifically promoting the expression of the biomarker.

The invention provides a pharmaceutical composition for treating hepatocellular carcinoma, which comprises an inhibitor of RP11-704M14.1 and/or RP11-498P 14.5; or an enhancer of HID1-AS1, wherein the inhibitor can inhibit the expression level of RP11-704M14.1 or RP11-498P 14.5; the promoter can increase the expression level of HID1-AS 1.

Further, the inhibitor is siRNA which specifically inhibits the biomarker.

Drawings

FIG. 1 is a graph showing the detection of the expression of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 genes in hepatocellular carcinoma tissues by QPCR, in which graph A shows RP11-704M14.1, graph B shows RP11-498P14.5, and graph C shows HID1-AS 1.

FIG. 2 is a ROC plot demonstrating diagnostic efficacy of different genes, where Panel A is RP11-704M14.1, Panel B is RP11-498P14.5, Panel C is HID1-AS1, Panel D is RP11-704M14.1 and RP11-498P14.5, Panel E is RP11-704M14.1 and HID1-AS1, Panel F is RP11-498P14.5 and HID1-AS1, Panel G is RP11-704M14.1 and RP11-498P14.5 and HID1-AS 1.

FIG. 3 is a graph of the effect of siRNA/overexpression vectors on the expression of RP11-704M14.1, RP11-498P14.5, HID1-AS1, where Panel A is RP11-704M14.1, Panel B is RP11-498P14.5, and Panel C is HID1-AS 1.

FIG. 4 is a graph showing the effects of RP11-704M14.1, RP11-498P14.5, and HID1-AS1 on hepatoma cell proliferation.

Detailed Description

The method screens lncRNA which presents differential expression in hepatocellular carcinoma tissues and corresponding paracarcinoma tissues by adopting a method combining high-throughput sequencing and bioinformatics, and then further verifies the relation between the lncRNA which is differentially expressed and hepatocellular carcinoma through a large sample, thereby determining the possibility of applying the lncRNA to diagnosis and treatment of hepatocellular carcinoma. Experiments show that the expression of RP11-704M14.1 and RP11-498P14.5 is up-regulated in liver cancer tissues for the first time, and the expression of HID1-AS1 is down-regulated in the liver cancer tissues.

The RP11-704M14.1 gene is located on human chromosome 4, and the RP11-704M14.1 gene in the invention comprises a wild type, a mutant type or a fragment thereof. The RP11-704M14.1 which has been disclosed so far has 2 transcripts, RP11-704M14.1-001 (transcript ID: ENST00000505646.1) and RP11-704M14.1-001 (transcript ID: ENST 00000504301.1). A representative RP11-704M14.1 gene is shown in the sequence of RP11-704M14.1-001 (transcription ID: ENST 00000505646.1).

The RP11-498P14.5 gene is located on human chromosome 9, and the RP11-498P14.5 of the invention comprises a wild type, a mutant type or a fragment thereof. There are 2 transcripts of RP11-498P14.5 which have been published so far, RP11-498P14.5-001 (transcription ID: ENST00000366109.2), RP11-498P14.5-002 (transcription ID: ENST 00000607322.1). A representative RP11-498P14.5 gene is shown in the sequence of RP11-498P14.5-001 (transcription ID: ENST 00000366109.2).

The HID1-AS1 gene is located on human chromosome 17, and the HID1-AS1 in the invention comprises a wild type, a mutant type or a fragment thereof. A representative HID1-AS1 gene is shown in the sequence of HID1-AS1-201 (transcription ID: ENST 00000577295.1).

It is known to those skilled in the art that when performing bioinformatic analysis on the original sequencing result, the sequencing result is usually aligned with a known gene, and the expression of the gene can be regarded as long as the sequenced fragment can be aligned with the related gene, so that when referring to a differentially expressed gene, different transcripts of the gene are also included in the present invention.

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. The present invention may utilize any method known in the art for determining gene expression.

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.

Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, In Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

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

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 referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein 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.beta.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 invention provides products for detecting the expression levels of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 genes, and the products comprise (but are not limited to) chips, nucleic acid membrane strips or kits.

Wherein, the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of sequences shown by RP11-704M14.1, RP11-498P14.5 and HID1-AS 1. 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.

The invention provides a nucleic acid membrane strip, which comprises a substrate and oligonucleotide probes which are fixed on the substrate and are used for RP11-704M14.1, RP11-498P14.5 and HID1-AS 1; 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 comprises a reagent for detecting the expression level of RP11-704M14.1, RP11-498P14.5 or HID1-AS1, and one or more substances selected from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme. The components of the kit may be packaged in aqueous medium or in lyophilized form.

Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial.

The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The invention provides application of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 in preparing a pharmaceutical composition for treating hepatocellular carcinoma, wherein the pharmaceutical composition comprises an inhibitor of RP11-704M14.1 and/or RP11-498P14.5 or an accelerant of HID1-AS 1;

and a pharmaceutically acceptable carrier.

The inhibitor is selected from interference molecules which take RP11-704M14.1, RP11-498P14.5 or transcripts thereof as target sequences and can inhibit the expression or gene transcription of RP11-704M14.1 and RP11-498P14.5 genes, and comprises: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

The promoter refers to any substance which can improve the stability of HID1-AS1 gene or expression product, up-regulate the expression of HID1-AS1, prolong the effective action time of IncRNA HID1-AS1 or promote the transcription of HID1-AS1 gene, and the substances can be used for the invention, and can be used AS substances which are useful for up-regulating the expression of HID1-AS1 gene, thereby being used for preventing or treating liver cancer.

The pharmaceutically acceptable carrier includes (but is not limited to) diluents, binders, surfactants, humectants, adsorptive carriers, lubricants, fillers, disintegrants.

The pharmaceutical compositions of the present invention may further comprise one or more anti-cancer agents. In a specific embodiment, the pharmaceutical composition comprises at least one compound that inhibits or promotes the expression of a biomarker 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.

The medicament of the invention can also be used in combination with other medicaments for treating liver cancer, and other therapeutic compounds can be simultaneously administered with the main active ingredients, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from 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. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with hepatocellular carcinoma

1. Sample collection

Cancer tissues and corresponding paracarcinoma tissue samples of 35 patients with primary hepatocellular carcinoma were collected, and 5 of the samples were randomly selected for high throughput sequencing. All patients in the group received no chemotherapy or radiation therapy prior to surgery.

2. Preparation and Mass analysis of RNA samples

Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein; then adding 200 mul of chloroform (chloroform), covering a pipe cover tightly, violently shaking for 15s, standing for 10min at normal temperature, centrifuging for 15min at the temperature of 4 ℃ and the rpm of 11000; transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after being reversed and mixed evenly, the mixture is stood for 10min at normal temperature, and then centrifuged for 15min at 11000rpm after being 4 ℃; carefully sucking away the liquid with a gun, leaving the precipitate at the bottom of the tube, adding 1ml of 75% ethanol, shaking on an oscillator for 5s, washing the precipitate once, and centrifuging at 4 ℃ and 8000rpm for 5 min; then carefully removing the supernatant, drying the precipitate for 10min, and adding a proper amount of water to dissolve the precipitate for 10 min.

3. Total RNA quantitation and purity analysis

The extracted RNA is subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA are detected by using Nanodrop2000, the integrity of the RNA is detected by the agarose gel electrophoresis, and the RIN value is determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

4. Construction of cDNA library

Ribosomal RNA was removed from total RNA using the Ribo-Zero kit from Epicentre; the complete RNA is randomly broken into small fragments of about 200bp by metal ions; construction of cDNA library was performed using the Truseq RNA sample Prep Kit from Illumina.

5. Sequencing

The cDNA library was sequenced using the Illumina X-Ten sequencing platform.

6. High throughput transcriptome sequencing data analysis

The non-detectable lncRNA was deleted and used for the bioassay using DESeq2 in tool R-3.3.3. Trim of 5 'and 3' segments of reads with cutadapt, trim off mass<20 bases and deleting reads with N greater than 10%; the sequencing results were then aligned to the reference genome using tophat. The reference genomic version used was hg 38; and (3) quantifying the expression quantity of the lncRNA by cuffquant and outputting the expression quantity in a standardized way, comparing the expression difference of lncRNA of a control group and a disease group by cuffdiff, and obtaining a screening standard for the lncRNA with the difference expression: FDR<0.05，abs(log₂FC)>1。

7. Results

The high-throughput sequencing result shows that the genes RP11-704M14.1, RP11-498P14.5 and HID1-AS1 show significant differential expression in hepatocellular carcinoma tissues, and the FDR is less than 0.05, wherein the genes RP11-704M14.1 and RP11-498P14.5 are significantly up-regulated in cancer tissues, and the genes HID1-AS1 are significantly down-regulated in cancer tissues, so that the potential application values of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 in the diagnosis of hepatocellular carcinoma are suggested.

Example 2 QPCR sequencing verification of differential expression of the RP11-704M14.1, RP11-498P14.5, HID1-AS1 genes

1. The gene differential expression of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 of 35 patients' liver cancer tissue samples and paracancerous tissue samples collected in the prior art are used for large-sample QPCR verification.

2. RNA extraction procedure see example 1

3. QPCR detection

1) Reverse transcription reaction

a. Mu.g of total RNA template was mixed with 2. mu.l of 10 Xbuffer, 2. mu.l of dATP (10mM), 0.5. mu.l of polyA polymerase, 0.5. mu.l of ribonuclease (RNase) inhibitor and ribonuclease free water (RNase free water) in a final volume of 20. mu.l and incubated at 37 ℃ for 1 h;

b. adding 1 μ l of 0.5 μ g/μ l Oligo (dT) -specific RT primer to the reaction tube, incubating at 70 ℃ for 5min, and immediately incubating on ice for at least 2 min;

c. the reaction mixture was mixed with 4. mu.l of 5 Xbuffer, 1. mu.l of dNTP (10mM), 0.5. mu. l M-MLV reverse transcriptase, 0.5. mu.l of RNase inhibitor, 10. mu.l of polyA reaction mixture and 4. mu.l of RNase free water, and incubated at 42 ℃ for 1 h.

2) Primer design

QPCR amplification primers are designed according to the coding sequences of RP11-704M14.1, RP11-498P14.5, HID1-AS1 gene and GAPDH gene, and the specific primer sequences are AS follows:

RP11-704M14.1 gene:

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

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

RP11-498P14.5 gene:

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

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

HID1-AS1 gene:

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

the reverse primer was 5'-TCCTTCTTCTCCTCCTCCTTCT-3' (SEQ ID NO. 6).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 7);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 8).

3) QPCR amplification assay

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, ddH₂O8.5. mu.l. All operations were performed on ice.

Reaction conditions are as follows: 10min at 95 ℃ (15 s at 95 ℃, 60 ℃ for 60) x 45 cycles. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, GAPDH is used as a reference gene, dissolution curve analysis is carried out at 60-95 ℃, a target band is determined by the dissolution curve analysis and electrophoresis, and relative quantification is carried out by a delta CT method.

4. Results

The QPCR results are shown in figure 1, compared with the tissues beside the cancer, the expression of RP11-704M14.1 and RP11-498P14.5 is obviously up-regulated in the liver cancer tissue, the expression of HID1-AS1 is obviously down-regulated in the liver cancer tissue, and the difference has statistical significance (P < 0.05).

Specifically, there were 38 samples showing significant up-regulation of RP11-704M14.1, 34 of which were cancer tissue samples, and 4 of which were paracancerous tissue samples; 36 samples with significant up-regulation of RP11-498P14.5, 33 of the cancer tissue samples and 3 of the paracarcinoma tissue samples; there were 35 samples exhibiting significant downregulation of HID1-AS1, 30 of cancer tissue samples and 5 of paracancerous tissue samples.

Example 3 validation of diagnostic efficacy of biomarkers

1. Data collection

The expression profile data of 371 liver cancer tissues and 50 paracarcinoma tissues are collected from a TCGA database, and the expression levels of lncRNA RP11-704M14.1, RP11-498P14.5 and HID1-AS1 in the liver cancer tissues and the paracarcinoma tissues are analyzed.

2. ROC curve analysis

The working characteristics of the subjects of lncRNA RP11-704M14.1, RP11-498P14.5 and HID1-AS1 are analyzed by using pROC package in R language, two accurate confidence spaces are calculated, and ROC curves are drawn.

3. Results

The ROC analysis results of candidate lncrnas are shown in table 1 and fig. 2, RP11-704M14.1(AUC ═ 0.851), RP11-498P14.5(AUC ═ 0.774), and HID1-AS1(AUC ═ 0.79) have higher area under the curve, which indicates that the diagnosis of hepatocellular carcinoma using RP11-704M14.1, RP11-498P14.5, and HID1-AS1 has higher accuracy and specificity. Meanwhile, ROC of the gene joint diagnosis is analyzed, and the result shows that the joint diagnosis of RP11-704M14.1 and RP11-498P14.5 has obviously increased AUC value and produces synergistic effect.

TABLE 1 AUC values of biomarkers as detection variables

Example 4 expression of biomarkers and their Effect on liver cancer cells

1. Cell culture

The hepatoma cell line HepG2 was purchased from ATCC. The cell lines were cultured in DMEM medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO₂Cultured in an incubator. Cell growth was observed daily and fluid changed every other day.

2. Transfection

1) Synthesis of siRNA

Interfering siRNAs against RP11-704M14.1 and RP11-498P14.5 were designed and synthesized by Shanghai Ji code pharmaceutical technology, Inc., and the control was general siRNA-NC. The sequences of relevant siRNAs are shown below:

siRNA-RP11-704M14.1：

the sense strand is 5'-UUCAUAGUCUUUCUUACAGCA-3' (SEQ ID NO.9)

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

siRNA-RP11-498P14.5：

The sense strand is 5'-UUGCUAUUUAUUUUAUCCCCU-3' (SEQ ID NO.11)

The antisense strand is 5'-GGGAUAAAAUAAAUAGCAAAG-3' (SEQ ID NO.12)

2) Construction of overexpression vectors

Specific PCR amplification primers are synthesized according to the sequence of HID1-AS1, and two restriction enzyme sites KpnI and XhoI are added to the 5 'end primer and the 3' end primer respectively. The cDNA sequence is double digested with restriction endonucleases KpnI and XhoI and inserted into eukaryotic cell expression vector pcDNA3.1(+) which is double digested with KpnI and XhoI, and the obtained recombinant vector pcDNA3.1(+) -HID1-AS1 is connected for subsequent experiment.

3) Transfection

Liposomes and OPTI-MEM reduced serum medium and siRNA/overexpression vectors and OPTI-MEM medium were mixed well and left at room temperature for 5min, respectively, according to the procedures of Lipofectamine 3000 kit instructions, and then liposomes, siRNA/overexpression vectors and OPTI-MEM medium were mixed and left at room temperature for 20 min. Adding the mixed solution into a serum-free cell culture medium, slightly shaking and uniformly mixing, and after incubation for 8 hours, changing the mixed solution into a complete culture medium containing 10% fetal calf serum to continue culture.

3. qRT-PCR detection of biomarker expression levels in cells

Total cellular RNA was extracted using Trizol method, reverse transcription and real-time quantitative PCR detection were performed as in example 1.

4. CCK-8 method for detecting cell proliferation activity

Taking cells in a logarithmic growth phase, carrying out heavy suspension counting, and inoculating 5000 cells/hole in a 96-well plate, wherein each group is provided with 5 multiple holes; culturing in incubator for 72 hr, adding CCK8 reagent, incubating at 37 deg.C in dark for 1 hr, detecting light absorption value (OD) with microplate reader at 450nm wavelength, and collecting data for statistical analysis.

5. Statistical analysis

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

6. Results

The silencing effect of siRNA is shown in FIGS. 3A and 3B, compared with the blank control group and the siRNA-NC group, the expression level of RP11-704M14.1/siRNA-RP11-498P14.5 of the group of transfected siRNA-RP11-704M 14.1/RP11-498P14.5 is obviously reduced, the difference is statistically significant (P <0.05), the effect of the over-expression vector is shown in FIG. 3C, compared with the blank control group and the transfected no-load group, the expression level of HID1-AS1 of the group of transfected pcDNA3.1(+) -HID1-AS1 is obviously increased, and the difference is statistically significant (P < 0.05).

The results of the effect of the biomarkers on the proliferation of hepatoma cells are shown in fig. 4, and the inhibition of the expression level of RP11-704M14.1/RP11-498P14.5 or the increase of the expression level of HID1-AS1 significantly reduces the proliferation activity of hepatoma cells, which indicates that RP11-704M14.1/RP11-498P14.5/HID1-AS1 plays an important role in the development of hepatoma, and can be used AS a molecular target for the treatment of hepatoma.

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

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Claims

1. The application of the reagent for detecting the expression level of the biomarker in preparing products for diagnosing hepatocellular carcinoma is characterized in that the biomarker selects one or more of RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

2. The use of claim 1, wherein the biomarkers are RP11-704M14.1 and RP11-498P 14.5.

3. The use of claim 1, wherein the product comprises reagents for detecting the expression level of the genes RP11-704M14.1, RP11-498P14.5, HID1-AS1 in a sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology.

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

probes specifically recognizing RP11-704M14.1, RP11-498P14.5 and HID1-AS 1; or

Primers for specifically amplifying RP11-704M14.1, RP11-498P14.5 and HID1-AS 1.

5. The application of claim 4, wherein the primer sequences for specific amplification of RP11-704M14.1, RP11-498P14.5 and HID1-AS1 are respectively shown in SEQ ID NO. 1-2, SEQ ID NO. 3-4 and SEQ ID NO. 5-6.

6. The use of claim 1, wherein the product comprises a chip, a kit, a nucleic acid membrane strip.

7. Application of an inhibitor of biomarkers RP11-704M14.1 and RP11-498P14.5 or a promoter of HID1-AS1 in preparing a pharmaceutical composition for treating hepatocellular carcinoma.

8. The use of claim 7, wherein the inhibitor is an siRNA that specifically inhibits a biomarker.

9. The use according to claim 8, wherein the promoter is a vector that specifically promotes the expression of a biomarker.