CN108707672B - Application of DUXAP8 in diagnosis and treatment of hepatocellular carcinoma - Google Patents

Abstract

The invention discloses application of DUXAP8 in diagnosis and treatment of hepatocellular carcinoma, and the invention firstly discovers that the expression of DUXAP8 is up-regulated in a hepatocellular carcinoma patient, and verifies that DUXAP8 can be used as a detection target to be applied to auxiliary diagnosis of hepatocellular carcinoma through a large sample. The invention also discovers that DUXAP8 is related to proliferation, migration and invasion of liver cancer cells, and prompts that DUXAP8 can be used as a drug target to be applied to treatment of hepatocellular carcinoma and hepatocellular carcinoma invasion and metastasis.

Description

Application of DUXAP8 in diagnosis and treatment of hepatocellular carcinoma

Technical Field

The invention belongs to the field of biological medicines, and relates to application of DUXAP8 in diagnosis and treatment of hepatocellular carcinoma.

Background

In recent years, hepatocellular carcinoma has become one of the most common malignant tumors in China, the mortality rate of which is the second of the most common malignant tumors, and seriously threatens the development of national economy and population health (Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global statistics. CA: a cancer J ournal for clinicians.2011,61(2): 69-90). Surgical resection, liver transplantation, radiotherapy and chemotherapy, interventional therapy, molecular targeted therapy and the like are the main treatment modes of liver cancer at present, but because liver cancer is hidden and has no obvious symptoms, factors such as the possibility of radical surgical treatment, postoperative recurrence and metastasis are lost when most patients visit the clinic, and the treatment effect and the survival time of liver cancer patients are seriously influenced (Ling TC, Kang JI, BushDA, Slater JD, Yang GY. protocol for hepatocellular cancer. Chinese patent outlet of cancer research.2012,24(4): 361-7.). Therefore, exploring and clarifying the exact mechanism of the occurrence, development and transfer of liver cancer cells on a molecular level, seeking new drug targets, and screening effective early diagnosis molecular markers are the hot spots of the current research and the important point of breaking through the bottleneck of liver cancer treatment.

With the completion of the Human Genome Project (HGP), new generation sequencing technologies have also been rapidly developed. With the development of high throughput sequencing technology, a large number of non-coding RNAs were annotated. The non-coding RNA is a kind of RNA which can not code protein, and the main function of the non-coding RNA is to regulate the transcription and expression of coding genes so as to influence the growth and development of organisms, the differentiation, proliferation, apoptosis, stress and other biological behaviors. The research of non-coding RNA is mainly focused on microRNA and long non-coding RNA (1 ncRNA). 1ncRNA is a non-coding RNA with the length of known transcript more than 200 nucleotides, and is involved in the occurrence and development of diseases through interaction with DNA, RNA and protein. Previous studies show that lncRNA is differentially expressed in liver cancer tissues, and the change of the expression level can inhibit the proliferation of liver cancer cells (CN201710404209.8 and CN 201710404203.0).

The research on lncRNA molecules in the liver cancer regulation mechanism is still in the initial stage at present. Therefore, the method plays an important role in diagnosis, prevention and treatment of the liver cancer by analyzing and researching the occurrence and development mechanism of the lncRNA in the liver cancer, and provides possibility for gene targeted treatment of the liver cancer.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a biomarker for early diagnosis or targeted therapy of hepatocellular carcinoma.

The second objective of the present invention is to provide a method for screening a candidate drug for treating hepatocellular carcinoma, which determines whether the substance to be screened is a candidate drug for treating hepatocellular carcinoma by detecting whether the substance to be screened can change the expression level of the marker.

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

the invention provides application of a reagent for detecting the expression level of DUXAP8 in preparation of a product for diagnosing hepatocellular carcinoma, wherein the expression level of DUXAP8 is up-regulated in a hepatocellular carcinoma patient.

Further, the agent is selected from:

a probe that specifically recognizes DUXAP 8; or

Primers for specific amplification of DUXAP 8.

Furthermore, the primer sequence of the specific amplification DUXAP8 is shown in SEQ ID NO. 3-4.

The invention provides a product for diagnosing hepatocellular carcinoma, which comprises a reagent for detecting the expression level of DUXAP8 in a sample. The product of the invention is not limited to common detection products such as chips, nucleic acid membrane strips, preparations or kits, as long as the product can detect the expression level of DUXAP 8. The "sample" includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, expectoration, alveolar bronchial lavage, urine, feces, etc. Preferably, the sample is tissue or blood.

Further, the reagents include a probe that specifically recognizes DUXAP8, or a primer that specifically amplifies DUXAP 8.

In a specific embodiment of the invention, the reagent comprises a primer for specifically amplifying DUXAP8, and the sequence of the primer for specifically amplifying DUXAP8 is shown as SEQ ID NO. 3-4.

The invention provides application of DUXAP8 gene in screening candidate drugs for treating hepatocellular carcinoma.

The invention provides a method for screening a candidate drug for treating hepatocellular carcinoma, which comprises the following steps:

treating a system expressing or containing the DUXAP8 gene with a substance to be screened; and

detecting the expression level of DUXAP8 gene in said system;

wherein, if the substance to be screened can reduce the expression level of the DUXAP8 gene (preferably significantly reduced, such as more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower), it indicates that the substance to be screened is a candidate drug for treating hepatocellular carcinoma.

The system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Such drug candidates include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds, and the like designed against the DUXAP8 gene or its upstream or downstream genes.

The present invention provides a composition comprising: an inhibitor of functional expression of DUXAP 8; and a pharmaceutically acceptable carrier. Wherein the inhibitor of functional expression of DUXAP8 is selected from: an interfering molecule targeting DUXAP8 or a transcript thereof and capable of inhibiting expression of a DUXAP8 gene or transcription of the gene, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

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

Preferably, the inhibitor is siRNA.

More preferably, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

The invention provides an application of DUXAP8 in preparing a pharmaceutical composition for treating hepatocellular carcinoma.

The invention provides application of DUXAP8 in preparing a pharmaceutical composition for treating hepatocellular carcinoma invasion.

The invention provides an application of DUXAP8 in preparing a pharmaceutical composition for treating hepatocellular carcinoma metastasis.

The invention provides application of a composition containing an inhibitor of DUXAP8 functional expression in preparing a medicament for treating hepatocellular carcinoma.

The invention provides application of a composition containing an inhibitor of DUXAP8 functional expression in preparing a medicament for treating hepatocellular carcinoma invasion.

The invention provides application of a composition containing an inhibitor of functional expression of DUXAP8 in preparing a medicament for treating hepatocellular carcinoma metastasis.

Drawings

FIG. 1 is a graph of the detection of expression of DUXAP8 in hepatocellular carcinoma patients using QPCR;

FIG. 2 is a graph showing the detection of the expression of DUXAP8 in hepatocellular carcinoma cells by QPCR;

FIG. 3 is a graph showing the effect of transfected siRNA on the expression of DUXAP8 in hepatocellular carcinoma cells;

FIG. 4 is a graph showing the effect of DUXAP8 on the proliferation of hepatoma cells using CCK 8;

FIG. 5 is a graph showing the effect of the DUXAP8 gene on migration of hepatocellular carcinoma cells;

FIG. 6 is a graph showing the effect of the DUXAP8 gene on hepatocellular carcinoma cell invasion.

Detailed Description

The invention is widely and deeply researched, the expression level of lncRNA in hepatocellular carcinoma tissues and tissues beside cancer is detected by a high-throughput sequencing method, lncRNA fragments with obvious expression difference are found, and the relation between the lncRNA fragments and the occurrence of hepatocellular carcinoma is discussed, so that a better way and a better method are found for early detection and targeted treatment of the hepatocellular carcinoma. Through screening, the invention discovers that DUXAP8 is remarkably up-regulated in hepatocellular carcinoma for the first time. Experiments prove that siRNA interference silences DUXAP8, can effectively inhibit proliferation of hepatocellular carcinoma cells, and provides a new way for personalized treatment of hepatocellular carcinoma.

DUXAP8 Gene

The DUXAP8 gene is located in region 1 of long arm 1 of chromosome 22, and the DUXAP8 in the present invention includes wild type, mutant or fragment thereof. In the examples of the present invention, the nucleotide sequence of a representative human DUXAP8 gene is shown in DUXAP8 gene (NR _122113.1) in GeneBank, the current International public nucleic acid database. The full-length sequence of the DUXAP8 nucleotide or a fragment thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. Two sequences are "substantially homologous" (or substantially similar) if, when the nucleic acid or fragment thereof is optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.

Alternatively, substantial homology or identity exists between nucleic acids or fragments thereof when the nucleic acids or fragments thereof hybridize to another nucleic acid (or the complementary strand thereof), one strand, or the complementary sequence thereof under selective hybridization conditions. Hybridization selectivity exists when hybridization is more selective than the overall loss of specificity. Typically, selective hybridization occurs when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90% identity over a stretch of at least about 14 nucleotides. As described herein, the length of the homology alignments can be a longer sequence segment, in certain embodiments generally at least about 20 nucleotides, more generally at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

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

Detection techniques

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

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

Generally, 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, RT-PCR uses Reverse Transcriptase (RT) to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA, TMA autocatalytically synthesizes multiple copies of the target nucleic acid sequence under substantially constant temperature, ionic strength, and pH conditions, wherein the multiple RNA copies of the target sequence autocatalytically generate additional copies, TMA optionally includes the use of a blocker, moiety, terminator, and other modifier to improve the sensitivity and accuracy of the TMA process, LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid, the DNA oligonucleotides are covalently linked by DNA ligase in repeated multiple cycles of thermal denaturation, hybridization, and ligation to produce detectable double-stranded ligated oligonucleotide products, SDA uses multiple cycles of primer sequences annealing to opposite strands of the target sequence, primer extension in the presence of dNTP α S to produce double-stranded half-stranded primer extension (phospho) primer extension, and displacement of the amplified primer sequences to produce a restriction endonuclease displacement product that mediates restriction of the displacement of the phosphomonoesters from the target nucleic acid sequences, and displacement of the amplified products.

Nucleic acid hybridization techniques of the invention 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.

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

Chip, nucleic acid membrane strip and kit

The chip in the invention comprises: a solid support; and oligonucleotide probes immobilized on the solid support in an ordered manner, the oligonucleotide probes specifically corresponding to part or all of the sequence set forth in DUXAP 8.

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

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

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

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

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

The present invention provides a kit that can be used to detect the expression of DUXAP 8. The kit comprises a specific primer pair for amplifying DUXAP 8; a standard DNA template; and (3) PCR reaction liquid. In a preferred embodiment, the specific primer pair comprises an upstream primer and a downstream primer, and the sequences are shown as SEQ ID NO. 3-4.

As a more preferable embodiment, the kit is a fluorescent quantitative PCR detection kit, and the primer is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes and composite probes.

In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.

In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg²⁺The fluorescent dye is SYBR Green II, and the Taq enzyme is hot start enzyme.

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 hepatocellular carcinoma) including the DUXAP8 gene, and can be used for simultaneously detecting a plurality of markers of the hepatocellular carcinoma, so that the accuracy of hepatocellular carcinoma diagnosis can be greatly improved.

Inhibitors and pharmaceutical compositions

Based on the inventors' findings, the present invention provides an inhibitor of DUXAP8, the properties of which are not important to the present invention, as long as it inhibits the functional expression of the DUXAP8 gene, and which is useful as a substance for down-regulating DUXAP8, for the prevention or treatment of hepatocellular carcinoma.

As a preferred mode of the invention, the inhibitor of DUXAP8 is a DUXAP 8-specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by respectively transfecting a hepatocellular carcinoma cell line by a transfection reagent, selects the siRNA with the best interference effect, and further performs experiments at a cellular level, and the result proves that the siRNA can effectively inhibit the expression level of DUXAP8 gene in cells and the proliferation of hepatocellular carcinoma cells.

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

As an alternative of the present invention, the inhibitor of DUXAP8 may also be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, the Small hairpin RNA being capable of inhibiting gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, the expression vector is a variety of vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The expression vector can be introduced into the host cell by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

The term "host cell" includes prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, such as a CHO cell, a COS cell, or the like.

The invention also provides a pharmaceutical composition comprising an effective amount of an inhibitor of the functional expression of DUXAP8, and a pharmaceutically acceptable carrier. The compositions are useful for inhibiting hepatocellular carcinoma. Any of the foregoing inhibitors of DUXAP8 may be used in the preparation of pharmaceutical compositions.

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

The present invention may employ various methods well known in the art for administering the inhibitor or gene encoding the inhibitor, or pharmaceutical composition thereof, to a mammal. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

Preferably, it can be carried out by means of gene therapy. For example, an inhibitor of DUXAP8 can be administered directly to a subject by a method such as injection; alternatively, expression units carrying inhibitors of DUXAP8 (e.g., expression vectors or viruses, etc., or siRNA or shRNA) can be delivered to the target site in a manner that results in the expression of the active DUXAP8 inhibitor, depending on the type of inhibitor, as is well known to those of skill in the art.

The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In particular embodiments, the pharmaceutical composition comprises at least one compound that inhibits the expression of DUXAP8 gene and at least one chemotherapeutic agent. Chemotherapeutic agents for use in the present invention include, but are not limited to: microtubule activators, alkylating agents, antineoplastic antimetabolites, platinum-based compounds, DNA-alkylating agents, antineoplastic antibiotic agents, antimetabolites, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial and exotoxin agents.

The pharmaceutical compositions of the invention may also be used in combination with other agents for the treatment of hepatocellular carcinoma, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

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

Statistical analysis

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

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with hepatocellular carcinoma

1. Sample collection

Cancer tissues and corresponding paracancerous tissue samples of 50 cases of HBV-infected hepatocellular carcinoma patients were collected, 8 cases were randomly selected for high throughput sequencing, and the patients gave their informed consent, and all the above samples 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. Mass analysis of RNA samples

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

4. Removal of rRNA

Ribosomal RNA was removed from total RNA using Ribo-Zero kit.

5. Construction of cDNA library

The construction of cDNA library was carried out using Illumina Truseq RNA sample Prep Kit, the specific procedures were as described in the specification.

6. Sequencing on machine

And (3) sequencing the cDNA library by using an Illumina X-Ten sequencing platform, wherein the specific operation is carried out according to the instruction.

7. High throughput transcriptome sequencing data analysis

The sequencing results were bioinformatically analyzed using DESeq2 in the R-3.3.3 kit, and differential analysis was performed by constructing dds matrix, normalization, screening criteria for differentially expressed lncRNA: FDR<0.05，abs(log₂FC)>2。

8. Results

The RNA-seq result shows that the expression level of the DUXAP8 gene in hepatocellular carcinoma tissues is remarkably higher than that of a control group, and the result suggests that the DUXAP8 can be used as a detection target for diagnosis of hepatocellular carcinoma.

Example 2 QPCR sequencing validation of differential expression of the DUXAP8 Gene

1. Large-sample QPCR validation of differential expression of DUXAP8 gene was performed using 50 patient cancer tissue samples and paracancerous tissue samples collected previously.

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

3. Reverse transcription:

1) 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.

2) Mu.l of 0.5. mu.g/. mu.l Oligo (dT) -specific RT primer was added to the reaction tube and incubated at 70 ℃ for 5 min.

3) Immediately incubated on ice for at least 2min to disrupt the secondary structure of the RNA and primers.

4) Mu.l of the above 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 ribonuclease (RNase) inhibitor, 10. mu.l of polyA reaction mixture and 4. mu.l of RNase free water, and incubated at 42 ℃ for 1 hour.

4. QPCR amplification assay

1) Designing a primer:

the primer sequence of housekeeping gene GAPDH is as follows:

a forward primer: 5'-CCGGGAAACTGTGGCGTGATGG-3' (SEQ ID NO.1)

Reverse primer: 5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3' (SEQ ID NO.2)

The primer sequence of the DUXAP8 gene is:

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

Reverse primer: 5'-CTGGATTCTGGACTCTTCTG-3' (SEQ ID NO.4)

2) 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. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

3) Reaction conditions

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, a target band is determined by melting curve analysis and electrophoresis, and relative quantification is carried out by a delta CT method.

5. Results

The results are shown in figure 1, the expression level of the DUXAP8 gene was up-regulated in hepatocellular carcinoma tissues compared to paracarcinoma tissues, the difference was statistically significant (P < 0.05); the expression of 48 patients is up-regulated, the expression of 1 patient is low, the expression of 1 patient is normal, and the positive detection rate is 48/50 × 100% and 96%; indicating that the DUXAP8 can be used as a detection index for diagnosing hepatocellular carcinoma.

Example 3 differential expression of the DUXAP8 Gene in hepatocellular carcinoma cell lines

1. Cell culture

Human hepatocellular carcinoma cell lines HepG2, Huh7 and normal hepatocyte 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

Total cellular RNA was extracted using a QIAGEN's cellular RNA extraction kit, and the specific procedures were as described in the instruction manual.

3. The reverse transcription was performed as in example 2

4. The specific steps of QPCR amplification assay were as in example 2

5. Results

As shown in FIG. 2, the expression of DUXAP8 gene was up-regulated in HepG2 and Huh7, compared with the normal liver cell line, and the difference was statistically significant (P <0.05), which is consistent with the result of RNA-sep.

Example 4 silencing of the DUXAP8 Gene

1. Cell culture

Human hepatocellular carcinoma cell line HepG2, 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. SiRNA design

siRNA sequence against DUXAP8 gene:

negative control siRNA sequence (siRNA-NC):

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

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

siRNA1：

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

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

siRNA2：

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

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

siRNA3：

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

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

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 according to Lipofectase transfection reagent 3000 (from Invitrogen) in DMEM Medium without double antibody and containing 10% FBSInstructions for transfection.

The experiments were 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 DUXAP8 gene at a concentration of 20 nM/well and was transfected separately at the same time.

3. QPCR detection of expression level of DUXAP8 Gene

3.1 extraction of Total RNA from cells the procedure was the same as in example 4.

3.2 reverse transcription procedure as in example 2.

3.3 QPCR amplification step as in example 2.

4. Results

Results as shown in fig. 3, the experimental group was able to reduce the expression level of DUXAP8, while the reduction level of siRNA1 group was the most significant, compared to HepG2, transfected empty siRNA-NC group, so siRNA1 was selected for the subsequent experiments.

Example 5 CCK8 assay for cell proliferation

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

2. CCK8 detection of cell proliferation

HepG2 cells in logarithmic proliferation phase were seeded in 96-well plates at 2X 10 per well³Dividing the cells into three groups, namely a blank control group, a transfection siRNA-NC group and a transfection siRNA1, wherein each group is provided with 6 multiple holes; after transfection for 0h, 24h, 48h, 72h, 108h and 120h, 10 mul/well of CCK8 reagent is added, and the mixture is put into an incubator to be incubated for 1h, and then the absorbance value of A450 is detected by using a microplate reader.

3. Results

The results shown in fig. 4 show that: the blank control group has no obvious difference with the unloaded group, while the transfected siRNA1 group has obviously lower cell growth rate than the control group, the difference has statistical significance (P <0.05), and the result shows that DUXAP8 is related to the proliferation of the liver cancer cells.

Example 6 cell migration assay

1. 24h after each group of cells are transfected, the cells are digested and centrifuged conventionally, and are resuspended by serum-free DMEM culture solution containing 10g/L, and the cell concentration is adjusted to be 1 multiplied by 10⁵/ml；

2. Inoculating 200 mu 1 of cell suspension into the upper chamber of a Transwell chamber without laying Matrigel gel;

3. adding 1ml of DMEM culture solution containing 10% fetal calf serum into the lower chamber of the 24-pore plate, and placing the Transwell chamber into the 24-pore plate to avoid air bubbles between the culture solution and the chamber; 37 ℃ and 5% CO₂Culturing for 48h by a conventional method;

4. taking out the small chamber, rinsing with PBS, carefully wiping off cells on the upper chamber surface of the small chamber with a cotton swab, fixing the lower chamber surface with methanol for 15min, and dyeing with 1% crystal violet for 5 min; 10 fields were randomly selected under a 200-fold inverted microscope, the number of cells passing through the lower layer of the microporous membrane was counted, and the average was taken, 3 cells per group, and repeated 3 times.

5. Results

As shown in FIG. 5, after transfection of interfering RNA into hepatocellular carcinoma cells, the migration capacity of the experimental group was significantly reduced compared to the control group, and the result indicates that DUXAP8 is involved in the migration process of hepatoma cells.

Example 7 cell invasion assay

1. Coating a basement membrane: putting the Matrigel gel in a refrigerator at 4 ℃ for overnight liquefaction, diluting the Matrigel in serum-free DMEM culture solution by using a precooled gun head on ice according to the proportion of 1:3, uniformly covering the serum-free DMEM culture solution on a Transwell cell membrane according to 50 mul/hole, and naturally air-drying at room temperature;

2. aspirating the residual liquid from the plate, adding 50. mu.l of serum-free medium containing L0g/L BSA to each well, and incubating at 37 ℃ for 30 min;

3. 24h after transfection of each group of cells, the cells were routinely digested, centrifuged, and resuspended in serum-free DMEM medium containing 10g/L BSA. Adjusting the cell concentration to 1 × l0⁵ml；

4. 200. mu.l of the cell suspension was inoculated into the upper chamber of a Transwell chamber coated with Matrigel gel; adding 1ml of DMEM culture solution containing 10% fetal calf serum into the lower chamber of the 24-pore plate, and placing the Transwell chamber into the 24-pore plate to avoid air bubbles formed between the culture solution and the chamber; 37 ℃ and 5% CO₂Culturing for 48h by a conventional method;

5. taking out the small chamber, leaching with PBS, carefully wiping the Matrigel glue and cells on the upper surface of the small chamber with a cotton swab, fixing the lower surface of the small chamber with methanol for 15min, and dyeing with 1% crystal violet for 5 min; rinse 2 times with PBS, randomly pick 10 fields under a 200-fold inverted microscope, count the number of cells that pass through the microporous membrane underlayer, average 3 chambers per group, and repeat 3 times.

6. Results

The results are shown in fig. 6, and the number of the invaded cells was decreased in the experimental group compared to the control group, indicating that DUXAP8 was related to the invasion of liver cancer cells.

Example 8 Effect of the DUXAP8 Gene on apoptosis of hepatocellular carcinoma cells

The effect of the DUXAP8 gene on apoptosis was examined using flow cytometry.

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

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

3. Apoptosis detection

1) 3m 110 Xloading buffer was diluted with 27ml of distilled water;

2) collecting a cell sample, washing the cell sample by using precooled PBS (phosphate buffer solution), adding the cells into lml 1 multiplied by sample loading buffer solution, centrifuging the cell sample for 10min at 300g, and sucking out the buffer solution;

3) the cell concentration in the cell suspension was adjusted to 1X 10 by adding 1X loading buffer again⁶Per ml;

4) taking out 100 mu 1 of cell suspension, and adding the cell suspension into an EP tube; adding 5 μ l Annexin V FITC into an EP tube, mixing the liquid in the EP tube uniformly, and incubating for 10min at room temperature in a dark place;

5) continuously adding 5 mu 1PI dye solution, and keeping out of the sun for 5min at room temperature;

6) adding 500. mu.l of PBS solution, mixing gently, and detecting by an up-flow cytometer within 1 h.

4. As a result:

the results showed no significant change in the rate of apoptosis (P <0.05) in the experimental group compared to the control group, indicating that expression of DUXAP8 was not associated with apoptosis of cancer cells.

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

Sequence listing

<110> Beijing, the deep biometric information technology GmbH

Beijing coordination hospital of Chinese medical academy of sciences

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Claims (10)

1. Application of reagent for detecting expression level of DUXAP8 in preparation of product for diagnosing hepatocellular carcinoma is provided.

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

a probe that specifically recognizes DUXAP 8; or

Primers for specific amplification of DUXAP 8.

3. The use according to claim 2, wherein the primer sequence for specific amplification of DUXAP8 is represented by seq id No.3 to 4.

Application of DUXAP8 gene in screening candidate drugs for treating hepatocellular carcinoma.

5. Use of a composition for the manufacture of a medicament for the treatment of hepatocellular carcinoma, the composition comprising: an inhibitor of functional expression of DUXAP8, and a pharmaceutically acceptable carrier; the inhibitor is siRNA.

6. The use of claim 5, wherein the siRNA has a sequence as shown in SEQ ID No. 7-8.

7. Use of a composition for the manufacture of a medicament for the treatment of hepatocellular carcinoma invasion, said composition comprising: an inhibitor of functional expression of DUXAP8, and a pharmaceutically acceptable carrier; the inhibitor is siRNA.

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

9. Use of a composition for the manufacture of a medicament for the treatment of metastasis of hepatocellular carcinoma, the composition comprising: an inhibitor of functional expression of DUXAP8, and a pharmaceutically acceptable carrier; the inhibitor is siRNA.

10. The use of claim 9, wherein the siRNA has a sequence as shown in SEQ ID No. 7-8.

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