CN107267625A - Purposes of the lncRNA as biomarker in liver cancer diagnosis and treatment - Google Patents

Abstract

Purposes the invention discloses lncRNA as biomarker in liver cancer diagnosis and treatment, specific mark is LOC101927480.Present invention firstly discovers that LOC101927480 up-regulated expressions in liver cancer patient, point out LOC101927480 to be used for the diagnosis and treatment of liver cancer as biomarker.The present invention further demonstrate that the expression for suppressing LOC101927480 can significantly reduce the propagation and Clone formation colony number of liver cancer cells by experiment, the new way that LOC101927480 expression is likely to become liver cancer treatment is intervened in prompting, while the present invention provides theoretical foundation for the research of liver cancer mechanism.

Description

The use of lncRNA as a biomarker in the diagnosis and treatment of liver cancer

technical field

The invention belongs to the field of biomedicine, and relates to the use of lncRNA as a biomarker in the diagnosis and treatment of liver cancer, in particular to the biomarker LOC101927480.

Background technique

Primary liver cancer is one of the most common malignant tumors in the world, its incidence ranks sixth among all cancers, and its mortality rate ranks second among all cancers. According to pathological classification, primary liver cancer can be divided into different types such as hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, and mixed hepatocellular carcinoma-intrahepatic cholangiocarcinoma. More than 90% of primary liver cancers are hepatocellular carcinomas. The occurrence of liver cancer is a multi-step and complicated process, mostly based on the chronic inflammation of the liver and the changed liver microenvironment, the mutation of many oncogenes or tumor suppressor genes, especially the gradual changes in their expression, and eventually induce liver cancer. Because in the early stage of liver cancer, most patients do not have typical clinical symptoms and signs, and it is difficult to find them clinically. Once typical symptoms appear, most of them are middle- and late-stage liver cancer. At this time, liver cancer is often accompanied by extrahepatic metastasis. There is no effective treatment for HCC cells disseminated throughout the body. With the progress of modern cell and molecular biology, it has been proved that the formation of liver cancer is often accompanied by a series of abnormalities in molecules and signaling pathways. Therefore, exploring the mechanism of liver cancer occurrence and development from the molecular level and using characteristic molecules as anti-tumor targets can open up new ways for clinical prevention and treatment of liver cancer.

With the development of high-throughput sequencing technology, the results of whole genome and transcriptome sequencing of liver cancer have shown that liver cancer has significant heterogeneity in molecular characteristics. For example, there are only mutations in as few as 5 genes in some liver cancer samples, while some There are as many as 121 gene mutations in HCC, but no ubiquitous gene mutation has been found in HCC. Therefore, the regulation of gene expression, especially epigenetic modification, such as DNA methylation, histone modification and abnormal expression of non-coding RNA, is worthy of in-depth study on the regulation of many differentially expressed genes in liver cancer.

Long noncoding RNA (long noncoding RNA, 1ncRNA) is a kind of RNA molecule with a transcript length of more than 200nt and no protein coding ability. Compared with protein coding sequences and microRNA, the research on lncRNA is still in its infancy, and its function needs to be further elucidated. Recent studies have shown that in many pathological conditions, including tumors, the expression level of lncRNAs changes, and lncRNAs can have multiple functions in physiological and pathological conditions, such as regulating cell proliferation, apoptosis, cell cycle, cell migration, etc. lncRNAs are involved in many important signal transduction regulatory processes such as genome imprinting, chromatin modification, transcriptional regulation, mRNA stability, protein translation regulation, microRNA function regulation, and protein function regulation. Therefore, studying the role of lncRNA in liver cancer is of great significance for revealing the molecular mechanism of liver cancer and the treatment of liver cancer.

Contents of the invention

In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide a product for diagnosing early liver cancer, so that patients can be treated at an early stage, thereby improving the survival rate and quality of life.

The second object of the present invention is to provide a treatment method and a pharmaceutical composition to realize precise molecular therapy of liver cancer.

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

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

The invention provides an application of a reagent for detecting LOC101927480 in preparing a product for diagnosing liver cancer. The reagents include reagents for RT-PCR, real-time quantitative PCR, in situ hybridization, northern blotting, chips or high-throughput sequencing platforms to detect the expression level of LOC101927480.

Further, the reagent is selected from:

A probe that specifically recognizes LOC101927480; or

Primers for specific amplification of LOC101927480.

Further, the primer sequences for specifically amplifying LOC101927480 are shown in SEQ ID NO.2 and SEQ ID NO.3.

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

Further, the product includes a chip, a preparation or a kit; wherein, the chip includes a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, and the oligonucleotide probe includes a The oligonucleotide probe against LOC101927480 level; the kit includes primers or chips for detecting the transcription level of LOC101927480.

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

"Probe" refers to a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe capable of binding to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, a probe is capable of binding a target polynucleotide that lacks complete sequence complementarity to the probe. Probes can be directly or indirectly labeled, including primers. Hybridization formats, 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 so-called "complementary" is not necessary as long as it is a hybrid. These polynucleotides generally have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% homology with the specific base sequence. These probes may be DNA or RNA, and in addition, a part or all of the nucleotides may be cross-linked by PNA (Polyamide nucleic acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Nucleic acid), ENA (registered trademark, 2′-O,4′-C-Ethylene-bridged nucleic acids), GNA (Glycerolnucleic acid, glycerol nucleic acid), TNA (Threose nucleic acid, threose nucleic acid) and other artificial nucleic acid replacement polynucleotide.

The gene detection kit or gene chip in the present invention can be used to detect the expression levels of multiple genes (for example, multiple genes related to liver cancer) including the LOC101927480 gene, and multiple markers of liver cancer can be detected at the same time, which can be greatly improved. Improve the accuracy of liver cancer diagnosis.

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

Further, the method of using LOC101927480 to screen potential substances for the prevention or treatment of liver cancer includes:

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

Detect 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 lower than 20%, preferably lower than 50%; more preferably lower than 80%), it indicates that the candidate substance is preventing or Potential substance for the treatment of liver cancer. The system is selected from: cell system, subcellular system, solution system, tissue system, organ system or animal system.

The candidate substances include (but are not limited to): interference molecules, nucleic acid inhibitors, small molecule compounds, etc. designed for the LOC101927480 gene or its upstream or downstream genes.

The invention provides the application of LOC101927480 in the preparation of a pharmaceutical composition for treating liver cancer.

Further, the pharmaceutical composition includes an inhibitor of the functional expression of LOC101927480, and the inhibitor can inhibit the expression of LOC101927480 or substances involved in the upstream or downstream pathway of LOC101927480.

Further, the inhibitor is siRNA against LOC101927480.

In the present invention, siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as RNAs that are different from naturally occurring RNAs by adding, deleting, replacing, and/or changing one or more nucleotides. The RNA is different from the altered RNA. Such alterations may include the addition of non-nucleotide material to, for example, 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 screening effective siRNA sequences, the present invention finds out the best effective fragment through a large number of comparison analysis. In a specific embodiment of the present invention, the inventors designed and synthesized a variety of siRNA sequences, and verified them by transfecting liver cancer cell lines with transfection reagents. As a result, interference molecules with better interference effects were detected. The sequences shown in SEQ ID NO.6 and SEQ ID NO.7 were further tested at the cell level, and the results proved that the inhibition efficiency was very high for cell experiments.

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

The present invention provides a pharmaceutical composition for treating liver cancer. The pharmaceutical composition includes an inhibitor of the functional expression of LOC101927480, and the inhibitor can act at the level of DNA or RNA (ie gene product).

Further, the pharmaceutical composition also includes other drugs compatible with the inhibitor of the functional expression of LOC101927480, as well as pharmaceutically acceptable carriers and/or adjuvants.

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

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

The medicine of the present invention may also include pharmaceutically acceptable coating materials including (but not limited to), rapidly decomposing coating materials, dyes, enteric polymers, plasticizers, water-soluble polymers, water-insoluble polymers, Dyes, pigments, other disintegrating agents. Common fast-disintegrating coating materials include OPADRY; enteric polymers include methacrylic acid polymers, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate, hydroxypropylmethylcellulose succinate, hydroxymethyl ethyl cellulose, cellulose acetylphosphophthalate; plasticizers include polyethylene glycol (PEG), propylene glycol, etc.

The pharmaceutical composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via implanted The medicine storage device administers medicine. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carriers, adjuvants or excipients. In certain instances, pharmaceutically acceptable acids, bases or buffers are used to adjust the pH of the formulation to increase the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intralesional, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the pharmaceutical composition of the present invention can be administered to the recipient through any route.

The pharmaceutical composition of the present invention can be orally administered in any oral dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. A lubricating agent, such as magnesium stearate, is typically also added. For oral administration in a 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 an oily phase and combined with emulsifying and/or suspending agents. If desired, some sweetening and/or flavoring and/or coloring agents may be added. Dosage unit formulations for oral administration can, where appropriate, be microencapsulated. The formulations can also be prepared to have extended or sustained release, for example, by coating or entrapping the particulate material in polymers, waxes or the like. The pharmaceutical composition of the present invention can be used to reduce the overexpression of endogenous LOC101927480, thereby treating liver cancer caused by the upregulation of LOC101927480 expression by reducing the expression of LOC101927480.

In the present invention, the compound that inhibits the expression of LOC101927480 can be administered to the subject as naked RNA together with a delivery agent as a nucleic acid (such as a recombinant plasmid or a virus vector), and the nucleic acid includes a sequence that inhibits the expression of LOC101927480. Delivery agents can be lipophilic agents, polycations, liposomes, and the like.

The medicine of the present invention can also be used in combination with other medicines for treating liver cancer, and other therapeutic compounds can be administered simultaneously with the main active ingredients, even in the same composition. The other therapeutic compounds may also be administered alone, in separate compositions or dosage forms different from the principal active ingredient. Some doses of the principal ingredient may be administered concurrently with other therapeutic compounds, while other doses may be administered alone. During the course of treatment, the dose of the pharmaceutical composition of the present invention can be adjusted according to the severity of symptoms, the frequency of relapses and the physiological response to the treatment regimen.

The pharmaceutical compositions of the present invention can 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 with a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. Liposomes can increase the blood half-life of gene products or nucleic acids. Suitable liposomes for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol such as cholesterol. Lipid selection is generally guided by consideration of factors such as the size of the liposome of interest and the half-life in the bloodstream.

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

The pharmaceutical composition of the present invention can be administered locally, and can be formulated into ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil .

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" in the present invention refers to an amount sufficient for a reasonable benefit/risk ratio applicable to medical treatment or prevention. The amount for treating or preventing diseases can be determined according to the severity of the disease, the activity of the drug, the patient's age, body weight, health, sex, the sensitivity of the patient to the drug, the administration time of the composition of the present invention used, the administration The effective dosage level is determined by the route and excretion rate, treatment time, the factors of the drugs used in combination with or used simultaneously with the composition of the present invention and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. In addition, single or multiple administrations can be performed. It is important to take all of the above factors into consideration and to administer an amount that can achieve the greatest effect with the least amount without side effects.

Those 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 by SEQ ID NO.1.

In the present invention, "functional expression" with respect to LOC101927480 means transcription and/or translation of a functional gene product. For non-protein-coding genes like LOC101927480, "functional expression" can be dysregulated on at least two levels. First, at the DNA level, for example by deletion or disruption of the gene, or if no transcription takes place (in both cases preventing the synthesis of the relevant gene product). Loss of transcription can result, for example, from epigenetic changes (eg DNA methylation) or from loss-of-function mutations. . A "loss of function" or "LOF" mutation as used herein 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 on the protein. Functional deletions can be caused by a wide range of mutation types, including but not limited to deletions of entire genes or portions of genes, splice site mutations, frameshift mutations caused by small insertions and deletions, nonsense mutations, missense substitutions for essential amino acids mutations, and mutations that prevent the correct cellular localization of the product. The definition also includes mutations in the promoter or regulatory regions of the LOC101927480 gene if those mutations interfere with the gene's function. Null mutations are LOF mutations that completely disrupt the function of a gene product. A null mutation in one allele will typically reduce expression levels by 50%, but can have serious effects on the function of the gene product. Notably, functional expression can also be dysregulated by gain-of-function mutations: by conferring a new activity on the protein, the normal function of the protein is dysregulated, and less functionally active protein is expressed. Vice versa, functional expression can be increased eg by gene duplication or by lack of DNA methylation. Functional expression can also be dysregulated by gain-of-function mutations: by conferring a new activity on the protein, the normal function of the protein is dysregulated, and less functionally active protein is expressed. Vice versa, functional expression can be increased eg by gene duplication or by lack of DNA methylation.

Second, at the RNA level, eg, through lack of efficient translation—eg because of mRNA instability (eg, through UTR variants), can result in mRNA being degraded before the transcript is translated. Or by lack of efficient transcription, for example because mutations induce novel splice variants.

In the present invention, the term "treatment" refers not to cure, but to slow down (reduce) the targeted pathological condition or disorder or prevent recurrence. If a patient is successfully "treated" after receiving a therapeutically effective amount of a therapeutic agent, the patient exhibits an observable and/or measurable reduction or disappearance of one or more of the signs and symptoms of a particular disease. For example, the number of cancer cells is significantly reduced or cancer cells disappear, reducing tumor size; inhibiting (i.e., to some extent slowing down, and preferably stopping) tumor metastasis; to some extent inhibiting tumor growth; to some extent reducing and and/or increased time to alleviate one or more symptoms associated with a particular cancer; reduced morbidity and mortality, and improved quality of life. Reduction of signs or symptoms of disease can be perceived by the patient. Treatment may result in a complete response - defined as disappearance of all signs of cancer, or a partial response - reduction in tumor size, preferably greater than 50%, more preferably 75%. Patients were also considered treated if they felt their disease was stable.

In a specific embodiment of the present invention, the experiment is all completed according to repeating at least 3 times, and the result data are all expressed in the form of mean ± standard deviation, and SPSS18.0 statistical software is used to carry out statistical analysis, both The difference between them was tested by t test, and it was considered to be statistically significant when P<0.05.

Description of drawings

Figure 1 shows the detection of the expression of LOC101927480 in liver cancer patients by QPCR;

Figure 2 shows the detection of the expression of LOC101927480 in liver cancer cells by QPCR;

Fig. 3 is a figure showing the influence of transfection siRNA on the expression of LOC101927480 in liver cancer cells detected by QPCR;

Figure 4 is a diagram of the influence of LOC101927480 on cell proliferation detected by CCK8;

Fig. 5 is a graph showing the effect of LOC101927480 on the clonogenic colony formation of cells.

specific implementation

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the embodiment, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Example 1 Screening Gene Markers Related to Liver Cancer

1. Sample collection

The cancer tissues and paracancerous tissues of 10 patients with liver cancer were collected, and the patients gave informed consent. All the above specimens were obtained with the consent of the organizational ethics committee.

2. Preparation of RNA samples

Tissue RNA was extracted using QIAGEN tissue RNA extraction kit, and the operation was performed according to the specific steps in the manual.

3. Reverse transcription and labeling

The 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

The gene chip uses Cannes Bio-Human lncRNA Array, and hybridization is performed according to the steps of the chip instruction manual.

5. Data processing

After hybridization, the chip was scanned with an Agilent scanner with a resolution of 5 μm. The scanner automatically scanned once at 100% and 10% PMT, and the results of the two times were automatically merged by Agilent software. The scanned image data was processed and analyzed using Feature Extraction, and the obtained raw data was processed using the Bioconductor program package. The final Ratio value is the experimental group and the control group. Screening criteria for differential genes: FDR<0.01, abs(log ₂ FC)>1.5.

6. Results

Compared with para-cancerous tissues, the expression level of LOC101927480 was significantly upregulated in HCC tissues.

Example 2 QPCR sequencing verification of differential expression of LOC101927480 gene

1. Large-scale QPCR verification of the differential expression of the LOC101927480 gene. According to the sample collection method in Example 1, 60 samples of liver cancer tissues and 60 samples of paracancerous tissues were collected.

2. The RNA extraction steps are the same as in Example 1.

3. Reverse transcription:

Use 25μl reaction system, take 1μg total RNA for each sample as template RNA, and add the following components to PCR tubes: DEPC water, 5× reverse transcription buffer, 10mM dNTP, 0.1mM DTT, 30μM Oligo dT, 200U/μl M-MLV, template RNA. Incubate at 42°C for 1h, then centrifuge briefly at 72°C for 10min.

(3) QPCR amplification test

Primer design:

The primer sequence of LOC101927480 gene is:

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

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

The primer sequence of the housekeeping gene GAPDH is:

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

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

A 25 μl reaction system was used, three parallel tubes were set up for each sample, and all amplification reactions were repeated more than three times to ensure the reliability of the results.

Prepare the following reaction system: SYBR Green polymerase chain reaction system 12.5 μl, forward and reverse primers (5 μM) 1 μl each, template cDNA 2.0 μl, enzyme-free water 8.5 μl. All operations were performed on ice.

The amplification program was: 95°C for 60s, (95°C for 15s, 60°C for 15s, 72°C for 45s)×35 cycles.

Using SYBR Green as a fluorescent marker, the PCR reaction was carried out on a Light Cycler fluorescence real-time quantitative PCR instrument, the target band was determined by melting curve analysis and electrophoresis, and the relative quantification was carried out by the ΔΔCT method.

3. Results

The results are shown in Figure 1. Compared with the paracancerous tissues, the expression of LOC101927480 gene was upregulated in liver cancer tissues, and the difference was statistically significant (P<0.05), which was consistent with the results of RNA-sep.

Example 3 Differential expression of LOC101927480 gene in liver cancer cell lines

1. Cell culture

Human liver cancer cell lines HepG2, Huh7 and normal liver cell line HL-7702 were cultured in DMEM containing 10% fetal bovine serum and 1% P/S in an incubator at 37°C, 5% CO ₂ , and 90% relative humidity. cultivated in. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.

2. Extraction of RNA

1) Stop the culture when the cells reach 80-90% confluency, digest with 0.25% trypsin and collect the cells in 1.5ml EP tubes, add lm1Trizol to each tube and shake slowly to break the cells, place on ice for 10min.

2) Remove protein and DNA: add 0.2ml chloroform to each 1.5ml EP tube, shake for 15s, and place at room temperature for 10min. Centrifuge at 12000rpm for 15min at 4°C.

The rest of the operation steps are the same as the RNA extraction process from the tissue.

3. Reverse transcription

Concrete steps are with embodiment 2.

4. Results

The results are shown in Figure 2. Compared with normal liver cell lines, the expression of LOC101927480 gene was up-regulated in liver cancer cells HepG2 and Huh7, and the difference was statistically significant (P<0.05), which was consistent with the results of RNA-sep.

Example 4 Silencing of the LOC101927480 gene

1. Cell culture

Human liver cancer cell line HepG2 was cultured in DMEM containing 10% fetal bovine serum and 1% P/S in an incubator at 37°C, 5% CO ₂ , and 90% relative humidity. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.

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)

Cells were seeded into a six-well cell culture plate at 2×10 ⁵ /well, and cultured in a 37°C, 5% CO ₂ incubator for 24 hours;

In DMEM medium without double antibody and containing 10% FBS, the transfection was performed according to the instructions of Lipofectamine Transfection Reagent 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). The siRNA of the negative control group had no homology with the sequence of the LOC101927480 gene, and the concentration was 20nM /well, while transfection was performed separately.

3. QPCR detection of expression level of LOC101927480 gene

3.1 Extraction of total cellular RNA

Concrete steps are with embodiment 3.

3.2 The steps of reverse transcription are the same as in Example 2.

3.3QPCR amplification steps are the same as in Example 2.

4. Statistical methods

The experiments were repeated 3 times, and the result data were expressed in the form of mean ± standard deviation. SPSS18.0 statistical software was used for statistical analysis to interfere with the difference between the LOC101927480 gene expression group and the control group Using t test, it is considered statistically significant when P<0.05.

5. Results

The results are shown in Figure 3. Compared with the HepG2, transfected empty siRNA-NC, siRNA2, and siRNA3 groups, the siRNA1 group can significantly reduce the expression of LOC101927480, and the difference is statistically significant (P<0.05).

Example 5 CCK8 detection cell proliferation experiment

1. Cell culture and transfection steps are the same as in Example 4

2. CCK8 detects cell proliferation

1) HepG2 cells in the logarithmic growth phase were seeded in a 96-well plate, with 2×10 ³ cells per well;

2) The experiment was divided into three groups, namely blank control group, siRNA-NC transfection group and siRNA2-LOC101927480 transfection group, with 6 replicate wells in each group;

3) Add 10 μl/well CCK8 reagent 24h, 48h, and 72h after transfection;

4) After 2 hours, use a microplate reader to detect the absorbance of A450.

3. Results

The results are shown in Figure 4: there was no significant difference between the blank control group and the empty load group, but the cell growth rate of the transfected siRNA1 group was significantly lower than that of the control group, and the difference was statistically significant (P<0.05). The above results showed that The expression of LOC101927480 can promote the growth of liver cancer cells.

Example 6 Soft agar colony formation experiment

1. Digest the cells in the logarithmic growth phase with 0.25% trypsin, pipette gently to make a single cell suspension, and collect the cell pellet by centrifugation.

2. Resuspend with DMEM complete medium containing 20% fetal bovine serum, count after appropriate dilution, and adjust the cell concentration to 5×10 ³ cells/ml.

3. Prepare two low-melting-point agarose solutions with concentrations of 1.2% and 0.7%, respectively, and maintain them in a 40°C water bath after autoclaving.

4. Mix 1.2% agarose and 2×DMEM medium 1:1, add 2×antibiotics and 20% calf serum, take 3ml of the mixed solution and pour it into a 6cm-diameter plate, let it cool and solidify for 5 minutes, and place it as the bottom agar in CO ₂ Reserve in the incubator.

5. Mix 0.7% agarose and 2×DMEM medium 1:1 in a sterile test tube, then add 0.2ml of stable infected cell suspension with a concentration of 5× ¹⁰³ cells/ml into the tube, mix well, Inject into the above-mentioned plate, gradually form a double agar layer, and repeat 4 samples in each experimental group.

6. After the upper layer of agar is solidified, place it in a 37°C, 5% _CO2 incubator for culture, and add 1.5ml of medium every 3 days.

7. After culturing for 14 days, take out the culture dish, and stain with 1 ml of 0.005% gentian violet for 90 minutes. Place the plate under an inverted microscope for observation, randomly select 10 low-power fields of view for each group of cells, and count the number of cell clones formed under the microscope.

8. Results

The results are shown in Figure 5, compared with the control group, the colony formation number of single cell clones in the cell group transfected with siRNA1 was significantly reduced.

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

SEQUENCE LISTING

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<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 cguguaaugg 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 (10)

1. detect application of the LOC101927480 reagent in the product of diagnosing liver cancer is prepared.

2. application according to claim 1, it is characterised in that the reagent is selected from：

Specific recognition LOC101927480 probe；Or

Specific amplification LOC101927480 primer.

3. application according to claim 3, it is characterised in that the primer sequence of the specific amplification LOC101927480 As shown in SEQ ID NO.2 and SEQ ID NO.3.

4. a kind of product of diagnosing liver cancer, it is characterised in that the product includes LOC101927480 expression water in detection sample Flat reagent.

5. product according to claim 5, it is characterised in that the product includes chip, preparation or kit.

6.LOC101927480 the application in screening prevention or the treatment potential material of liver cancer.

7.LOC101927480 the application in the pharmaceutical composition for preparing treatment liver cancer.

8. application according to claim 7, it is characterised in that described pharmaceutical composition includes LOC101927480 features The inhibitor of expression.

9. a kind of pharmaceutical composition for treating liver cancer, it is characterised in that described pharmaceutical composition includes LOC101927480 functions Property expression inhibitor.

10. pharmaceutical composition according to claim 9, it is characterised in that described pharmaceutical composition also includes and the suppression Other compatible medicine classes of preparation and pharmaceutically acceptable carrier and/or auxiliary material.