CN106868183B

CN106868183B - Application of WFDC21P in diagnosis and treatment of liver cancer

Info

Publication number: CN106868183B
Application number: CN201710213759.1A
Authority: CN
Inventors: 王冬国; 陈佳玉; 杨林军
Original assignee: Taizhou Municipal Hospital
Current assignee: Taizhou Municipal Hospital
Priority date: 2017-04-01
Filing date: 2017-04-01
Publication date: 2020-03-13
Anticipated expiration: 2037-04-01
Also published as: CN106868183A

Abstract

The invention discloses application of WFDC21P in diagnosis and treatment of liver cancer. The invention discovers for the first time that the expression of WFDC21P gene in liver cancer patients is up-regulated, and the change of the transcription level of WFDC21P can change the proliferation and cloning formation number of liver cancer cells, and prompts that WFDC21P can be used for developing products for early diagnosis of liver cancer and medicines for treating liver cancer. The invention also provides a theoretical basis for the research of the molecular mechanism of the liver cancer.

Description

Application of WFDC21P in diagnosis and treatment of liver cancer

Technical Field

The invention belongs to the field of biological medicines, and relates to application of WFDC21P in diagnosis and treatment of liver cancer.

Background

Liver cancer is one of the most common malignant tumors and one of the major causes of death due to tumors worldwide. 80% of new liver cancer cases occur in developing countries, and hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in China. Although local treatments such as surgery and transcatheter arterial chemoembolization can treat liver cancer, patients with liver cancer still have a high rate of recurrence and metastasis. The molecular mechanisms of HCC recurrence or metastasis are still not well understood at present. Epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play important roles in the progression of cancer metastasis. In particular, it has been found that EMT may be a significant cause of acquired resistance during metastatic dissemination and treatment of tumors. Recent data also indicate that epigenetic changes are also involved in the metastatic process of cancer.

Recently, it has been found that in addition to the existence of microRNA, a micro non-coding RNA with strong regulation and epigenetic modification functions, there are tens of thousands of other non-coding RNAs, namely long non-coding RNAs (lncRNAs), long non-coding RNA, which are RNA molecules with transcript length exceeding 200nt and have no function of coding protein in the human genome. lncRNA was originally thought to be a byproduct of RNA polymerase II transcription, is "noise, waste" of genome transcription, and has no biological function. However, recent studies have shown that they can affect the expression level of genes at various levels through a variety of different mechanisms such as DNA methylation, histone modification, post-transcriptional regulation, RNA interference, imprinting of genes, and the like. Therefore, lncRNA may become a new direction for liver cancer research. In the prior art, microRNA has been reported to play an important role in liver cancer development and drug resistance, but the biological function and the role of the lncRNA serving as a non-coding product in the liver cancer development process are unclear, and further research is needed.

At present, lncRNA which is abnormally expressed in tumor tissues can relate to various systems of the whole body and is distributed widely, but because of the huge number of lncRNA, the research aiming at lncRNA in the field of cancer is still in the initial stage, so that the research on molecular basis of LncRNA liver cancer has important significance.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides a diagnostic product, which provides a basis for early diagnosis of liver cancer.

The second objective of the invention is to provide a molecular marker for clinical diagnosis and treatment of liver cancer and mechanism research.

The third objective of the invention is to provide a therapeutic means and a pharmaceutical composition, which can realize precise molecular therapy of liver cancer by reducing the transcription level of target molecules.

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

the invention provides application of a reagent for detecting the transcription level of WFDC21P in preparing a product for diagnosing liver cancer, wherein the transcription level of WFDC21P is up-regulated in a liver cancer patient.

Further, the agent is selected from:

a probe that specifically recognizes WFDC 21P; or

Primers for specific amplification of WFDC 21P.

The invention provides a product for diagnosing liver cancer, which can diagnose liver cancer by detecting the transcription level of WFDC21P in a sample. 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 product comprises a chip, a preparation or a kit; wherein, the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, and the oligonucleotide probes comprise oligonucleotide probes aiming at WFDC21P and used for detecting the transcription level of WFDC 21P; the gene detection kit comprises a reagent for detecting the transcription level of WFDC 21P.

The invention provides application of WFDC21P gene in screening potential substances for preventing or treating liver cancer.

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

treating a system expressing or containing WFDC21P gene or with a candidate substance; and

detecting transcription of WFDC21P gene in the system;

wherein, if the candidate substance can reduce the transcription level of WFDC21P gene (preferably significantly reduced, such as more than 20%, preferably more than 50%, more preferably more than 80%), it indicates that 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 WFDC21P gene or its upstream or downstream gene.

The invention provides application of WFDC21P in preparing a pharmaceutical composition for treating liver cancer.

Further, the pharmaceutical composition comprises an inhibitor of functional expression of WFDC21P selected from the group consisting of: an interfering molecule targeting WFDC21P or its transcript and capable of inhibiting WFDC21P gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. Preferably, the inhibitor is siRNA.

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

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

an inhibitor of functional expression of WFDC 21P; and

a pharmaceutically acceptable carrier.

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

Drawings

FIG. 1 is a graph showing the expression of WFDC21P in liver cancer patients by QPCR;

FIG. 2 is a graph showing the expression of WFDC21P in liver cancer cells by QPCR;

FIG. 3 is a graph showing the detection of transfection of transfected WFDC21P in liver cancer cells by QPCR;

FIG. 4 is a graph showing the effect of WFDC21P on the proliferation of hepatoma cells measured using CCK 8;

FIG. 5 is a graph showing the effect of WFDC21P expression on the proliferation potency of hepatoma cells, as measured by soft agar colony formation assay.

Detailed Description

The invention is widely and deeply researched, the transcription level of lncRNA in a liver cancer specimen in a liver cancer patient and a healthy person is detected by adopting an lncRNA chip which covers the most database at present through a high-throughput method, lncRNA fragments with obvious expression difference are found, and the relation between the lncRNA fragments and the occurrence of liver cancer is discussed, so that a better way and a better method are found for the early detection and the targeted treatment of the liver cancer. Through screening, the invention discovers that WFDC21P is remarkably upregulated in liver cancer for the first time. Experiments prove that siRNA interference silences WFDC21P, can effectively inhibit the proliferation of liver cancer cells, and provides a new approach for personalized treatment of liver cancer.

WFDC21P gene

WFDC21P gene is located on No.3 band of No. 17 chromosome long arm 2 region, 8 transcripts are present in the gene at present, and the nucleotide sequence of a representative human WFDC21P gene is shown in SEQ ID NO. 1. WFDC21P in the present invention includes wild type, mutant type or fragments thereof.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. 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.

Thus, the polynucleotide of the invention preferably has at least 75%, more preferably at least 85%, more preferably at least 90% homology with SEQ ID NO. 1. More preferably, there is at least 95%, more preferably at least 98% homology.

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

Detection techniques

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

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

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

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

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

Chip and kit

The chip in the invention comprises: a solid support; and oligonucleotide probes immobilized on the solid phase carrier in order, and the oligonucleotide probes specifically correspond to partial or whole sequence of WFDC 21P.

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).

The present invention provides a kit which can be used to detect expression of WFDC 21P. Preferably, the preparation or the kit further comprises a marker for marking the RNA sample, and a substrate corresponding to the marker. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit also comprises an instruction manual and/or chip image analysis software.

The gene detection kit or gene chip can be used for detecting the transcription levels of a plurality of genes (for example, a plurality of genes related to liver cancer) including WFDC21P gene, and can simultaneously detect a plurality of markers of liver cancer, thereby greatly improving the accuracy of liver cancer diagnosis.

Inhibitors and pharmaceutical compositions

Based on the findings of the inventors, the present invention provides an inhibitor of WFDC21P, the properties of which are not important to the present invention, as long as it inhibits the functional expression of WFDC21P gene, and these inhibitors are useful as a substance for down-regulating WFDC21P, and useful for preventing or treating liver cancer.

In a preferred embodiment of the present invention, the inhibitor of WFDC21P is a small interfering RNA molecule specific for WFDC 21P. 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 transfecting a liver cancer cell line with a transfection reagent respectively, selects the siRNA with the best interference effect, and further performs a cell level experiment, and the result proves that the siRNA can effectively inhibit the transcription level of WFDC21P gene in cells and the proliferation of liver cancer cells.

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

The invention also provides a pharmaceutical composition, which contains an effective amount of the inhibitor of WFDC21P and a pharmaceutically acceptable carrier. The composition can be used for inhibiting liver cancer. Any of the foregoing inhibitors of WFDC21P 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 WFDC21P can be administered directly to a subject by a method such as injection; alternatively, expression units carrying inhibitors of WFDC21P (e.g., expression vectors or viruses, etc., or siRNA or shRNA) can be delivered to the target in a manner that results in the expression of active WFDC21P 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 a specific embodiment, the pharmaceutical composition comprises at least one compound that inhibits the expression of WFDC21P gene and at least one chemotherapeutic agent. Chemotherapeutic agents for use in the present invention include, but are not limited to: microtubule activators, alkylating agents, antineoplastic antimetabolites, platinum-based compounds, DNA-alkylating agents, antineoplastic antibiotic agents, antimetabolites, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial and exotoxin agents.

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

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

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

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of Gene markers associated with liver cancer

1. Sample collection

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

2. Preparation of RNA samples

Tissue RNA extraction was performed using a tissue RNA extraction kit from QIAGEN, and the procedures were performed according to the specific procedures described in the specification.

3. Reverse transcription and labelling

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

4. Hybridization of

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

5. Data processing

After hybridization, the chip was scanned with an Agilent scanner with a resolution of 5 μm, the scanner automatically scanned 1 time each with 100% and 10% PMT, and the results of 2 Agilent software were automatically merged. And (3) processing and analyzing the scanned image data by adopting Feature Extraction, and performing subsequent data processing on the obtained original data by applying a Bioconductor program package. The final Ratio values are experimental and control. Differential gene screening criteria: the up-regulated gene is the ratio more than or equal to 4, and the down-regulated gene is the ratio less than or equal to 0.25.

6. Results

WFDC21P transcript levels in liver cancer tissues significantly higher than in paracarcinoma tissues compared to paracarcinoma tissues.

Example 2QPCR sequencing validation of differential expression of WFDC21P Gene

1. Large sample QPCR validation was performed on WFDC21P 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 WFDC21P gene is:

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

Reverse primer: 5'-ATTATGTCTCTGGGTCTT-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 cDNA 2.0. 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. Statistical method

The experiments were performed in 3 replicates, the results were represented as mean ± sd, and were statistically analyzed using SPSS18.0 statistical software, with the difference between the two using the t-test, and considered statistically significant when P < 0.05.

4. Results

As shown in FIG. 1, WFDC21P gene was transcriptionally up-regulated in liver cancer tissues compared to the paracancerous tissues, and the differences were statistically significant (P <0.05), consistent with the RNA-sep results.

Example 3 differential expression of WFDC21P Gene in liver cancer cell lines

1. Cell culture

Human liver cancer cell strain HepG2,Huh7 and Normal liver cell line HL-7702, in DMEM medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

2. Extraction of RNA

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

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

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

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

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

3. Reverse transcription

The specific procedure is the same as in example 2.

4. Statistical method

5. Results

As shown in FIG. 2, WFDC21P gene was up-regulated in liver cancer cells HepG2 and Huh7 compared with normal liver cell line, and the difference was statistically significant (P <0.05), consistent with the result of RNA-sep.

Example 4 silencing of WFDC21P Gene

1. Cell culture

Human hepatoma cell line HepG2 in DMEM medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 5% CO₂And culturing in an incubator with relative humidity of 90%. Changing the solution for 1 time 2-3 days, and performing conventional digestion with 0.25% trypsin containing EDTAAnd (4) generation.

2. SiRNA design

siRNA sequence against WFDC21P gene:

negative control siRNA sequence (siRNA-NC):

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

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

siRNA1-WFDC21P：

sense strand: 5'-UCAACUUCAGUCAAUAGAGGU-3' (SEQ ID NO.8),

antisense strand: 5'-CUCUAUUGACUGAAGUUGACA-3' (SEQ ID NO. 9);

siRNA2-WFDC21P：

sense strand: 5'-AUCCUUAAGAUAACUCUUCAU-3' (SEQ ID NO.10),

antisense strand: 5'-GAAGAGUUAUCUUAAGGAUCA-3' (SEQ ID NO. 11);

siRNA3-WFDC21P：

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

the antisense strand is 5'-CAUCUUUCCCUAAGAUCGUCA-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-WFDC21P, siRNA2-WFDC21P, siRNA3-WFDC21P), wherein the siRNA of the negative control group had no homology with the sequence of WFDC21P gene and had a concentration of 20 nM/well, and transfection was performed separately.

3. QPCR detection of transcript level of WFDC21P 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 results were expressed as mean ± sd, and the statistical analysis was performed using SPSS18.0 statistical software, and the difference between the interfering WFDC21P gene expression group and the control group was considered statistically significant when P <0.05 using t-test.

5. Results

The results are shown in fig. 3, compared with HepG2, transfected unloaded siRNA-NC, siRNA1-WFDC21P, siRNA3-WFDC21P group, siRNA2-WFDC21P group can significantly reduce the expression of WFDC21P, and the difference is statistically significant (P < 0.05).

Example 5CCK8 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-WFDC21P, wherein each group is provided with 6 multiple holes;

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

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

3. Statistical method

The experiments were performed in 3 replicates using SPSS13.0 statistical software for statistical analysis, and the differences between the two were considered statistically significant when P <0.05 using the t-test.

4. Results

The results shown in fig. 4 show that: the blank control group has no obvious difference with the unloaded group, while the transfected siRNA2-WFDC21P group has obviously lower cell growth rate than the control group, the difference has statistical significance (P <0.05), and the result shows that the expression of WFDC21P can promote the growth of the liver cancer 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, put in 5% CO at 37 DEG C₂The 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 siRNA2-WFDC 21P-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> WFDC21P in diagnosis and treatment of liver cancer

<160>13

<170>PatentIn version 3.5

<210>1

<211>630

<212>DNA

<213> human source

<400>1

gggggaagaa gaaaagagga tgccagtcct tgttcctagg ccaagacctg agccctgtaa 60

tattaatatt tggatctaaa atcagccttc caacagcaac atgaagttgg cagccttcct 120

cctcctgtga tcctcatcat cttcagccta gaggtacaag agcttcaggc tgcaggagac 180

cggcttttgg gtgtctgacc cttctcaaca cagcatcaga cagccacctc tattgactga 240

agttgacaat ggagaataaa actcattacc cctgctgaat ccaggggccc ctttttacag 300

atgagaaaag ttccagaaag acccagagac ataatcaggt acctgcgtcg agctctgcac 360

aggtgactgg gactgcaacc ccggagacca ctgtgtcagc aatgggtgtg gccatgagtg 420

tgttgcagga tgaagagtta tcttaaggat catctttccc taagatcgtc atcccttcct 480

ggagttccta tcttccaaga tgtgactgtc tggagttcct tgactaggaa gatggatgaa 540

aacagcaagc ctgtggatgg agactacagg ggatatggga ggcagggaag aggggttgtt 600

tcttttaata aatcatcatt gttaaaagca 630

<210>2

<211>19

<212>DNA

<213> Artificial sequence

<400>2

tctattgact gaagttgac 19

<210>3

<211>18

<212>DNA

<213> Artificial sequence

<400>3

attatgtctc tgggtctt 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

ucaacuucag ucaauagagg u 21

<210>9

<211>21

<212>RNA

<213> Artificial sequence

<400>9

cucuauugac ugaaguugac a 21

<210>10

<211>21

<212>RNA

<213> Artificial sequence

<400>10

auccuuaaga uaacucuuca u 21

<210>11

<211>21

<212>RNA

<213> Artificial sequence

<400>11

gaagaguuau cuuaaggauc a 21

<210>12

<211>21

<212>RNA

<213> Artificial sequence

<400>12

acgaucuuag ggaaagauga u 21

<210>13

<211>21

<212>RNA

<213> Artificial sequence

<400>13

caucuuuccc uaagaucguc a 21

Claims

1. Application of a reagent for detecting WFDC21P transcription level in preparing 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 that specifically recognizes WFDC 21P; or

Primers for specific amplification of WFDC 21P.

3. A product for diagnosing liver cancer, which diagnoses liver cancer by detecting the transcription level of WFDC21P in a sample.

4. The product of claim 3, wherein the product comprises a chip or a kit.

Application of WFDC21P gene in screening potential substances for preventing or treating liver cancer.

6. A method for screening a potential substance for preventing or treating liver cancer, comprising:

treating a system expressing or containing WFDC21P gene with a candidate substance; and

detecting transcription of WFDC21P gene in the system;

wherein, if the candidate substance can reduce the transcription level of WFDC21P gene, the candidate substance is a potential substance for preventing or treating liver cancer.

Application of an inhibitor of WFDC21P functional expression in preparing a pharmaceutical composition for treating liver cancer.

8. The use of claim 7, wherein the pharmaceutical composition further comprises other drugs compatible with the inhibitor and pharmaceutically acceptable carriers and/or excipients.

9. A pharmaceutical composition for treating liver cancer, comprising:

an inhibitor of functional expression of WFDC 21P; and

a pharmaceutically acceptable carrier.