CN107267625B - Application of lncRNA as biomarker in liver cancer diagnosis and treatment - Google Patents

Abstract

The invention discloses application of lncRNA as a biomarker in diagnosis and treatment of liver cancer, and particularly discloses LOC101927480 as the biomarker. The invention discovers that LOC101927480 is up-regulated in liver cancer patients for the first time, and prompts that LOC101927480 can be used as a biomarker for diagnosis and treatment of liver cancer. Experiments further prove that the inhibition of the expression of LOC101927480 can obviously reduce the proliferation of liver cancer cells and the colony forming number by cloning, and the intervention of the expression of LOC101927480 is prompted to be a new way for treating liver cancer, and meanwhile, the invention provides a theoretical basis for the research of a liver cancer mechanism.

Description

Application of lncRNA as biomarker in liver cancer diagnosis and treatment

Technical Field

The invention belongs to the field of biomedicine, relates to application of lncRNA as a biomarker in diagnosis and treatment of liver cancer, and particularly relates to a biomarker LOC 101927480.

Background

Primary liver cancer is one of the most common malignant tumors worldwide, with the incidence of the sixth and the mortality of the second. According to pathological typing, primary liver cancer can be classified into different types, such as hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma, and hepatocellular carcinoma-intrahepatic cholangiocellular carcinoma mixed type. More than 90% of primary liver cancers are hepatocellular carcinomas. The occurrence of liver cancer is a multi-step and complex process, and most of the liver cancer is induced by the mutation of a plurality of oncogenes or cancer suppressor genes, especially the gradual change of the expression of the oncogenes or the cancer suppressor genes on the basis of the existing chronic inflammation of the liver and the changed liver microenvironment. Because the early stage of liver cancer, patients mostly have no typical clinical symptoms and signs, and are difficult to find clinically, once the typical symptoms appear, the patients are mostly liver cancer of middle and late stages, the liver cancer is often accompanied by intrahepatic and extrahepatic metastasis of the liver cancer, and because liver cancer cells are insensitive to chemotherapy, no effective treatment method for the liver cancer cells which are scattered systemically exists at present. With the progress of modern cell molecular biology, it has been proved that liver cancer is formed with a series of molecular and signaling pathway abnormalities. Therefore, the mechanism of liver cancer generation and development is explored from the molecular level, and the characteristic molecules are used as anti-tumor targets, so that a new way for clinically preventing and treating liver cancer can be developed.

With the development of high-throughput sequencing technology, the sequencing result of the whole genome and transcriptome of liver cancer shows that liver cancer has significant molecular characteristic heterogeneity, and if some liver cancer samples only have mutations of as few as 5 genes, and some liver cancer samples also have mutations of as many as 121 genes, but no ubiquitous gene mutation is found in liver cancer. Therefore, the regulation of gene expression, especially epigenetic modification such as DNA methylation, histone modification and non-coding RNA expression, is worth further discussing the regulation of a plurality of differentially expressed genes in liver cancer.

Long non-coding RNA (1 ncRNA) is a RNA molecule with the transcript length of more than 200nt and without protein coding capacity. Compared with protein coding sequences and microRNAs, the research on lncRNA is still in the initial stage, and the functions of lncRNA are to be further clarified. Recent studies have shown that lncRNAs are expressed at altered levels in many pathological conditions, including tumors, and that lncRNAs can have multiple functions in physiological and pathological conditions, such as regulation of cell proliferation, apoptosis, cell cycle, cell migration, etc. The lncRNAs participate in a plurality of important signal transduction regulation processes such as genome imprinting, chromatin modification, transcription regulation, mRNA stability, protein translation regulation, microRNA function regulation, protein function regulation and the like. Therefore, the research on the function of lncRNA in liver cancer has important significance for disclosing the molecular mechanism of liver cancer pathogenesis and treating liver cancer.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a product for diagnosing early liver cancer, so that a patient can be treated at an early stage, and the survival rate and the life quality are improved.

The second purpose of the invention is to provide a treatment means and a pharmaceutical composition for realizing precise molecular therapy of liver cancer.

The third object of the present invention is to provide a method for screening a potential substance for preventing or treating liver cancer.

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

the invention provides an application of a reagent for detecting LOC101927480 in preparing a product for diagnosing liver cancer. The reagent comprises RT-PCR, real-time quantitative PCR, in-situ hybridization, northern blotting and reagents for detecting the expression level of LOC101927480 by a chip or a high-throughput sequencing platform.

Further, the agent is selected from:

a probe specifically recognizing LOC 101927480; or

A primer for specifically amplifying LOC 101927480.

Further, the primer sequence of the specific amplification LOC101927480 is shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention provides a product for diagnosing liver cancer, which comprises a reagent for detecting the expression level of LOC101927480 in a sample. The "sample" includes, but is not limited to, cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluids, expectoration, alveolar bronchial lavage, urine, stool, etc. Preferably, the sample is tissue or blood.

Further, the product comprises a chip, a preparation or a kit; wherein, the chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, and the oligonucleotide probes comprise oligonucleotide probes aiming at LOC101927480 and used for detecting LOC101927480 transcription level; the kit comprises a primer or a chip for detecting LOC101927480 transcription level.

The solid phase carrier 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.

"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.

The probe has 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).

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 LOC101927480 gene, and simultaneously detecting a plurality of markers of the liver cancer, thereby greatly improving the accuracy of liver cancer diagnosis.

The invention provides an application of LOC101927480 in screening potential substances for preventing or treating liver cancer.

Further, the method for screening potential substances for preventing or treating liver cancer by using LOC101927480 comprises the following steps:

treating a system expressing or containing the LOC101927480 gene with a candidate substance; and

detecting the expression of the LOC101927480 gene in the system;

wherein, if the candidate substance can reduce the expression level of the LOC101927480 gene (preferably significantly reduced, such as more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower), 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 against the LOC101927480 gene or its upstream or downstream genes.

The invention provides an application of LOC101927480 in preparing a pharmaceutical composition for treating liver cancer.

Further, the pharmaceutical composition comprises an inhibitor of functional expression of LOC101927480, said inhibitor being capable of inhibiting the expression of LOC101927480 or a substance involved in the upstream or downstream pathway of LOC 101927480.

Further, the inhibitor is an siRNA against LOC 101927480.

In the present invention, siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include adding non-nucleotide material, e.g., to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.

When the invention is used for screening effective siRNA sequences, the optimal effective segment is found out through a large amount of comparison analysis. In the specific implementation mode of the invention, the inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting a liver cancer cell line with a transfection reagent respectively, so that the result detects interference molecules with better interference effect, the interference molecules have the sequences shown in SEQ ID No.6 and SEQ ID No.7 respectively, and further the result of a cell level experiment proves that the inhibition efficiency is very high for a cell experiment.

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 present invention provides a pharmaceutical composition for the treatment of liver cancer comprising an inhibitor of LOC101927480 functional expression, which may act at the DNA level or at the RNA (i.e. gene product) level.

Furthermore, the pharmaceutical composition also comprises other medicines which are compatible with the LOC101927480 functional expression inhibitor and pharmaceutically acceptable carriers and/or auxiliary materials.

Further, the vector includes (but is not limited to): diluents, excipients, binders, wetting agents, disintegrants, absorption enhancers, surfactants, humectants, adsorption carriers, lubricants, and the like.

In the present invention, the pharmaceutical composition may be prepared using various additives, such as buffers, stabilizers, bacteriostats, isotonic agents, chelating agents, pH controlling agents.

The medicaments of the present invention may also include pharmaceutically acceptable coating materials including, but not limited to, fast-dissolving coating materials, colorants, enteric polymers, plasticizers, water-soluble polymers, water-insoluble polymers, dyes, pigments, other disintegrating agents. Common fast-decomposing coating materials include OPADRY; enteric polymers include methyl internal olefine acid polymer, phosphorus hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate, hydroxypropyl methyl cellulose succinate, hydroxyethyl cellulose, acetyl phosphorus benzene two cellulose ester; plasticizers include polyethylene glycol (PEG), propylene glycol, and the like.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred.

The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, pharmaceutically acceptable acids, bases or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form in which it is administered. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracolic, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may be administered to a subject by any route as long as the target tissue is reached.

The pharmaceutical compositions of the present invention may be administered orally in any oral dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, carriers that are commonly used include lactose and corn starch. Lubricating agents such as magnesium stearate are also typically added. For oral administration in capsule form, suitable diluents include lactose and anhydrous corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in the oil phase and combined with emulsifying and/or suspending agents. If desired, sweetening and/or flavouring and/or colouring agents may be added. Dosage unit formulations for oral administration may be microencapsulated, as appropriate. The formulations may also be prepared to provide extended or sustained release, for example, by coating or embedding the particulate material in a polymer, wax, or the like. The pharmaceutical composition can be used for reducing endogenous LOC101927480 overexpression, and treating liver cancer caused by up-regulation of LOC101927480 expression by reducing the expression of LOC 101927480.

In the present invention, the compound inhibiting the expression of LOC101927480 may be administered to a subject as naked RNA together with a delivery agent as a nucleic acid (such as a recombinant plasmid or viral vector) comprising a sequence inhibiting the expression of LOC 101927480. The delivery agent may be a lipophilic agent, a polycation, a liposome, or the like.

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 pharmaceutical compositions of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Liposomes can increase the blood half-life of the gene product or nucleic acid. Suitable liposomes for use in the present invention can be formed from standard vesicle-forming lipids, which typically include neutral or negatively charged phospholipids and a sterol, such as cholesterol. In general, the choice of lipid is guided by taking into account factors such as the size of the liposome of interest and the immediate half-life in the bloodstream.

Liposomes for use in the present methods may comprise a ligand molecule that targets the liposome to a cell. Ligands that bind to receptors ubiquitous in cancer cells, such as monoclonal antibodies that bind to tumor cell antigens, are preferred. Liposomes useful in the present invention may also be modified to avoid clearance by the monocyte macrophage system and reticuloendothelial system. Such modified liposomes have opsonization-inhibiting moieties present on the surface or incorporated into the liposome structure. Preferably, the liposome may comprise both an opsonization-inhibiting moiety and a ligand.

The pharmaceutical composition of the present invention may be a pharmaceutical composition for topical administration, and may be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the age, body weight, health, sex, patient's sensitivity to the drug, administration time of the composition of the present invention to be used, administration route and excretion ratio, treatment time, factors of the drug to be used in combination or concomitantly with the composition of the present invention to be used, and other factors known in the medical field. The pharmaceutical composition of the present invention may be administered as a single therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, administration may be performed in a single or multiple doses. It is important to consider all of the above elements and to administer them in an amount that achieves the maximum effect with the minimum amount of side effects.

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 marker gene may have the cDNA sequence specified in SEQ ID NO. 1.

In the present invention, "functional expression" in relation to LOC101927480 means transcription and/or translation of a functional gene product. For non-protein encoding genes like LOC101927480, "functional expression" may 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 LOC101927480 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.

In the present invention, the term "treatment" means not aiming at a cure, but slowing (reducing) the targeted pathological condition or disorder or preventing a relapse. If a patient is successfully "treated" after receiving a therapeutically effective amount of a therapeutic agent, the patient exhibits observable and/or measurable reduction or disappearance of signs and symptoms of one or more particular diseases. For example, a significant reduction in the number of cancer cells or disappearance of cancer cells, reduction in tumor size; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; increasing the time to reduce to some extent and/or alleviate one or more symptoms associated with a particular cancer; reduced morbidity and mortality, and improved quality of life. The reduction in signs or symptoms of the disease can be perceived by the patient. Treatment may achieve a complete response-defined as the disappearance of all signs of cancer, or a partial response-a reduction in tumor size, preferably by a proportion of more than 50%, more preferably 75%. Patients are also considered treated if they experience stable disease.

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.

Drawings

FIG. 1 is a graph showing the expression of LOC101927480 in liver cancer patients by QPCR;

FIG. 2 is a graph showing the expression of LOC101927480 in liver cancer cells by QPCR;

FIG. 3 is a graph showing the effect of QPCR on the expression of LOC101927480 by transfected siRNA in hepatoma cells;

FIG. 4 is a graph showing the effect of LOC101927480 on cell proliferation using CCK 8;

FIG. 5 is a graph of the impact of LOC101927480 on colony formation by clones of cells.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. 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. The scanned image data is processed and analyzed by Feature Extraction, and the obtained original data is subjected to subsequent data processing by applying a Bioconductor program packageAnd (6) processing. The final Ratio values are experimental and control. Differential gene screening criteria: FDR<0.01，abs(log₂FC)>1.5。

6. Results

Compared with the tissues beside the cancer, the expression level of LOC101927480 in the liver cancer tissues is obviously up-regulated.

Example 2QPCR sequencing verification of differential expression of the LOC101927480 Gene

1. Large sample QPCR verification was performed on LOC101927480 gene differential expression. 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 from each sample as template RNA, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mM dNTP, 0.1mM DTT, 30. mu.M Oligo dT, 200U/. mu. l M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.

(3) QPCR amplification assay

Designing a primer:

the primer sequence of LOC101927480 gene is:

a forward primer: 5'-AATCTAGGACTTACGCTCTT-3' (SEQ ID NO.2)

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

The primer sequence of housekeeping gene GAPDH is as follows:

a forward primer: 5'-CCGGGAAACTGTGGCGTGATGG-3' (SEQ ID NO.4)

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

A25. mu.l reaction system was used, with 3 parallel channels per sample, and all amplification reactions were repeated three more times to ensure the reliability of the results.

The following reaction system was prepared: SYBR Green polymerase chain reaction system 12.5. mu.l, forward and reverse primers (5. mu.M) 1. mu.l each, template cDNA2.0. mu.l, 8.5. mu.l enzyme-free water. All operations were performed on ice.

The amplification procedure was: 95 ℃ 60s, (95 ℃ 15s, 60 ℃ 15s, 72 ℃ 45s) x 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, compared with the tissues beside the cancer, the expression of LOC101927480 gene is up-regulated in the liver cancer tissues, and the difference is statistically significant (P <0.05), which is consistent with the result of RNA-sep.

Example 3 differential expression of the LOC101927480 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) The culture was terminated when the cells reached 80-90% confluence, and the cells were harvested by 0.25% trypsinization in 1.5m1EP tubes, disrupted by adding lm1Trizol to each tube and left on ice for 10 min.

2) Deproteinization, DNA removal: 0.2ml of chloroform was added to each 1.5m1EP tube, shaken for 15 seconds, and allowed to stand at room temperature for 10 min. Centrifuge at 12000rpm for 15min at 4 ℃.

The remaining procedures were the same as in the extraction of RNA from tissues.

3. Reverse transcription

The specific procedure is the same as in example 2.

4. Results

As shown in FIG. 2, compared with the normal liver cell line, the LOC101927480 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 4 silencing of the LOC101927480 Gene

1. Cell culture

A human liver cancer cell strain HepG2,using DMEM 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

siRNA sequence against LOC101927480 gene:

negative control siRNA sequence (siRNA-NC):

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

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

siRNA1：

the sense strand is 5'-AUGUUGAUGGCGUGUAUGGUA-3' (SEQ ID NO.8),

the antisense strand is 5'-CCAUACACGCCAUCAACAUUU-3' (SEQ ID NO. 9);

siRNA2：

the sense strand is 5'-AAUAUCCUAUCUACAAGAGAC-3' (SEQ ID NO.10),

the antisense strand is 5'-CUCUUGUAGAUAGGAUAUUUU-3' (SEQ ID NO. 11);

siRNA3：

the sense strand is 5'-AGUAGUACAUUUCAAAGAGUC-3' (SEQ ID NO.12),

the antisense strand is 5'-CUCUUUGAAAUGUACUACUUC-3' (SEQ ID NO.13)

The cells were arranged 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 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 the LOC101927480 gene at a concentration of 20 nM/well, and was transfected separately.

3. QPCR detection of expression level of LOC101927480 gene

3.1 extraction of Total RNA from cells

The specific procedure is the same as in example 3.

3.2 reverse transcription procedure as in example 2.

3.3QPCR amplification step as in example 2.

4. Statistical method

The experiments were performed in 3 replicates, the data of the results were represented as mean ± standard deviation, statistically analyzed using SPSS18.0 statistical software, and the difference between the expression group of the interfering LOC101927480 gene and the control group was determined to be statistically significant when P <0.05 using t-test.

5. Results

The results are shown in fig. 3, compared with HepG2, transfection empty load siRNA-NC, siRNA2, siRNA3 group, siRNA1 group was able to significantly reduce the expression of LOC101927480, the difference was statistically significant (P < 0.05).

Example 5 CCK8 assay for cell proliferation

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

2. CCK8 detection of cell proliferation

1) HepG2 cells in logarithmic proliferation phase were seeded in 96-well plates at 2X 10 per well³(ii) individual cells;

2) the experiment is divided into three groups, namely a blank control group, a transfection siRNA-NC group and a transfection siRNA2-LOC101927480, and each group is provided with 6 multiple holes;

3) adding 10 mu l/well of CCK8 reagent after 24h, 48h and 72h of transfection respectively;

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

3. Results

The results are shown in FIG. 4: the blank control group has no obvious difference with the unloaded group, while 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 result shows that the expression of LOC101927480 can promote the growth of the hepatoma cells.

Example 6 Soft agar colony formation experiment

1. Cells in logarithmic phase were digested with 0.25% trypsin, gently pipetted to form a single cell suspension, and the cell pellet was collected by centrifugation.

2. Resuspending in DMEM complete medium containing 20% fetal calf serum, diluting properly, counting, adjusting cell concentration to 5 × 10³One per ml.

3. Two low melting point agarose solutions were prepared at 1.2% and 0.7% concentrations, respectively, and after autoclaving, were maintained in a 40 ℃ water bath.

4. Mixing 1.2% agarose and 2 × DMEM medium at a ratio of 1:1, adding 2 × antibiotic and 20% calf serum, adding 3ml mixed solution into a plate with diameter of 6cm, standing for 5min, cooling and solidifying, and placing in CO as bottom agar₂And 4, keeping the temperature in the incubator for later use.

5. 0.7% agarose and 2 × DMEM medium were mixed 1:1 in a sterile tube and 0.2ml 5 × 10 concentration added to the tube³Each/ml of stably infected cell suspension was mixed well and poured into the above dish to gradually form a layer of diisetron, with 4 replicates per experimental group.

6. After the upper agar is solidified, the mixture is placed at 37 ℃ in 5% CO₂The cells were incubated in an incubator with 1.5ml of medium every 3 days.

7. After 14 days of culture, the dish was removed and stained with 1ml of 0.005% gentian violet for 90 min. The plate was placed under an inverted microscope for observation, and 10 low power fields were randomly selected for each group of cells, and the number of cell clones formed by the under-the-lens technique was counted.

8. Results

As shown in FIG. 5, colony formation was significantly reduced in the single cell clone of the siRNA 1-transfected cell group compared to the control group.

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> Wang Dong nationality

Application of <120> lncRNA as biomarker in liver cancer diagnosis and treatment

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<170>PatentIn version 3.5

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<210>2

<211>20

<212>DNA

<213> Artificial sequence

<400>2

aatctaggac ttacgctctt 20

<210>3

<211>18

<212>DNA

<213> Artificial sequence

<400>3

cactgaatgg cttgtctg 18

<210>4

<211>22

<212>DNA

<213> Artificial sequence

<400>4

ccgggaaact gtggcgtgat gg 22

<210>5

<211>25

<212>DNA

<213> Artificial sequence

<400>5

aggtggagga gtgggtgtcg ctgtt 25

<210>6

<211>19

<212>RNA

<213> Artificial sequence

<400>6

uucuccgaac gugucacgu 19

<210>7

<211>19

<212>RNA

<213> Artificial sequence

<400>7

acgugacacg uucggagaa 19

<210>8

<211>21

<212>RNA

<213> Artificial sequence

<400>8

auguugaugg cguguauggu a 21

<210>9

<211>21

<212>RNA

<213> Artificial sequence

<400>9

ccauacacgc caucaacauu u 21

<210>10

<211>21

<212>RNA

<213> Artificial sequence

<400>10

aauauccuau cuacaagaga c 21

<210>11

<211>21

<212>RNA

<213> Artificial sequence

<400>11

cucuuguaga uaggauauuu u 21

<210>12

<211>21

<212>RNA

<213> Artificial sequence

<400>12

aguaguacau uucaaagagu c 21

<210>13

<211>21

<212>RNA

<213> Artificial sequence

<400>13

cucuuugaaa uguacuacuu c 21

Claims (7)

1. Application of a reagent for detecting LOC101927480 in preparation of a product for diagnosing liver cancer.

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

a probe specifically recognizing LOC 101927480; or

A primer for specifically amplifying LOC 101927480.

3. The use according to claim 2, characterized in that the primer sequence for specific amplification LOC101927480 is shown as SEQ ID No.2 and SEQ ID No. 3.

4. A product for diagnosing liver cancer, which is characterized by comprising a reagent for detecting the expression level of LOC101927480 in a sample, wherein the reagent comprises primers shown as SEQ ID NO.2 and SEQ ID NO. 3.

The application of an inhibitor of LOC101927480 functional expression in preparing a pharmaceutical composition for treating liver cancer is characterized in that the inhibitor is siRNA.

6. The pharmaceutical composition for treating liver cancer comprises an inhibitor of LOC101927480 functional expression, wherein the inhibitor is siRNA.

7. The pharmaceutical composition of claim 6, further comprising other drugs compatible with the inhibitor and a pharmaceutically acceptable carrier and/or adjuvant.

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