WO2021248587A1 - Marker for diagnosing liver cancer, detection reagent and application thereof - Google Patents

Abstract

The present invention provides a marker for diagnosing liver cancer. The marker is TRAF2. The expression of the TRAF2 in a liver cancer patient is upregulated.

Description

Marker for diagnosing liver cancer and detection reagent and application thereof Technical field

The invention belongs to the field of biomedicine, and is used for diagnosing liver cancer markers and detection reagents and applications thereof, and specifically relates to the application of TRAF2 in the diagnosis and treatment of liver cancer.

Background technique

Primary liver cancer (Primary liver cancer, PLC) is one of the few tumors whose incidence rate continues to increase. In the world, liver cancer ranks fourth among cancer-related deaths. China is a major liver cancer country. According to statistics from the World Health Organization, in 2018, there were 392,868 new liver cancer cases (9.2%) and 368,960 deaths (12.9%) in China. Both morbidity and mortality ranked third among all tumors. Although the diagnosis and treatment of liver cancer have made some progress, the onset of liver cancer is insidious, and the early signs are often atypical, and the disease is often in the stage of disease progression. Therefore, the incidence of liver cancer not only increases, but the overall survival rate of liver cancer patients is low. Therefore, studying the main molecular mechanisms of the early occurrence and development of human liver cancer and identifying new high-sensitivity and specific markers of liver cancer are also of far-reaching practical significance for the continuous search for new methods of molecular diagnosis and treatment of liver cancer.

In recent years, there have been more and more researches on the pathogenesis of primary liver cancer. The current research mainly focuses on mutations at the gene level and related signal pathways. Mutations in the promoter region of telomerase reverse transcriptase (TERT) are the most common genetic variation, accounting for about 60% of liver cancer mutations. In addition, the promoter regions of TP53, ARIDA1A, CTNNB1, and Axin1 are also common mutation regions. . Currently known signal pathways involved in the occurrence and development of primary liver cancer mainly include NF-κB pathway, autophagy pathway, PI3K/AKT/mTOR signal pathway and other signal pathways. These gene mutations and related signal pathways related to the occurrence and development of liver cancer Changes affect cell proliferation and apoptosis, cell differentiation and other biological processes, leading to disorders of normal physiological regulation and ultimately tumorigenesis. Therefore, by targeting oncogenes and key molecules in related signal pathways, it helps to prevent tumor progression at an early stage and provides new strategies for the treatment of liver cancer.

The tumor necrosis factor receptor-associated factor (TRAF) family is an important type of adaptor protein. Currently, the TRAF family has 7 members (TRAF1-TRAF7). A typical feature of the TRAF family is that they have a common conserved region (except TRAF7) at their C-terminus, that is, the TRAF domain. The TRAF domain mediates protein-protein interactions, not only can mediate the interaction of TRAF molecules with upstream and downstream pathway signal molecules, but also mediate the oligomerization of TRAF. Therefore, an important role of the TRAF family is to act as an adaptor protein in the assembly of receptor-related signal complexes, linking upstream receptors with downstream effector molecules. In addition, TRAF family members contain the RING structure, so they are also considered to have E3 ubiquitin ligase activity. Ubiquitinization modification is an important post-translational modification of proteins, which relies on the ubiquitin-proteasome system (UPS). UPS is a way of protein degradation in cells and is essential for maintaining protein homeostasis. Protein ubiquitination is a three-step enzymatic cascade that depends on ATP. First, ubiquitin activating enzyme E1 activates ubiquitin molecules (ubiquitin, Ub), and then the activated Ub is transferred to ubiquitin conjugating enzyme E2; finally, ubiquitin The ligase E3 recognizes and recruits the target protein and catalyzes the transfer of ubiquitin from E2 to the target protein. This process usually requires the covalent attachment of the ubiquitin molecule to the lysine residue (K) of the target protein. At present, TRAF2 has been proven to have ubiquitin E3 ligase activity.

Existing studies have shown that TRAF2 is abnormally amplified and rearranged in a variety of tumor tissues, and mutated in a variety of tumor tissues. The most common form of mutation is a missense mutation. TRAF2 participates in a variety of signals including NF-κB. The regulation of pathways regulates the proliferation and survival of tumor cells and other important biological processes. Therefore, in-depth exploration of the expression level of TRAF2 in liver cancer and its mechanism of action will lay the foundation for elucidating the molecular mechanism of liver cancer progression and provide potential for early diagnosis and treatment of liver cancer. The target.

Summary of the invention

The present invention provides the application of TRAF2 in the diagnosis and treatment of liver cancer. In order to achieve the above objectives, the present invention adopts the following technical solutions:

The present invention provides a marker for diagnosing liver cancer, the marker is TRAF2, and the expression of TRAF2 is up-regulated in patients with liver cancer.

The present invention provides the application of TRAF2 in preparing a pharmaceutical composition for preventing or treating liver cancer. Further, the pharmaceutical composition includes a TRAF2 functional expression inhibitor.

The invention provides the use of TRAF2 in screening drugs for diagnosis and treatment of liver cancer.

The present invention provides a reagent for detecting the expression level of the marker TRAF2, and the reagent includes the following primers:

TRAF2-F: 5'-GCCCTTCAACCAGAAGGTGAC-3’

TRAF2-R: 5'-CCAACCCCCAGACACCAGTA-3'.

The invention provides the use of a reagent for detecting the expression level of TRAF2 in preparing a kit for diagnosing liver cancer. The expression of TRAF2 is up-regulated in patients with liver cancer.

In addition, the present invention provides a preparation or kit containing a reaction reagent for detecting the expression level of TRAF2.

The third aspect of the present invention provides the application of the preparation or kit in the preparation of products for diagnosing liver cancer.

The beneficial effects achieved by the present invention:

The present invention detects the expression level of TRAF2 in clinical liver cancer tissue samples in detail, and finds that the mRNA and protein expression levels of TRAF2 in liver cancer tissues are significantly higher than those in adjacent tissues. Constructing a model of hepatocarcinoma cells with knockdown and overexpression of TRAF2, and found that TRAF2 can significantly promote the proliferation and cloning ability of hepatocarcinoma cells. The downstream targets of TRAF2 were screened. The immunoprecipitation experiment found that TRAF2 binds to p62 for the first time. Further ubiquitination analysis showed that TRAF2 can add non-degradable K63 polyubiquitin chains to p62 molecules. The nude mouse tumor formation experiment further confirmed that knocking out TRAF2 significantly inhibited the growth of liver cancer in the mouse subcutaneous tumor model and reduced the volume and quality of the tumor. These results all show that TRAF2 can play a significant role in promoting the growth of liver cancer through post-translational ubiquitination regulation of p62, and its significant high expression in liver cancer tissues may be a key factor involved in the occurrence and progression of liver cancer. The invention provides a new biomarker TRAF2 for the diagnosis of liver cancer, and TRAF2 can be used as a new target for the treatment of liver cancer, and provides a new direction for the screening of drugs for diagnosis and treatment of liver cancer and the treatment of liver cancer.

Description of the drawings

Figure 1 shows the detection of TRAF2 protein and mRNA expression levels in cancer tissues and adjacent tissues in clinical specimens of liver cancer patients.

Figure 2 shows the effect of knocking out TRAF2 on the proliferation and cloning ability of liver cancer cells.

Figure 3 shows the effect of knocking out TRAF2 on the ability of hepatocarcinoma cells to form subcutaneous tumors.

Figure 4 shows the effect of overexpression of TRAF2 on the proliferation and cloning ability of liver cancer cells.

Figure 5 shows that TRAF2 exerts a tumor suppressor effect by directly binding to p62

Figure 6 shows that TRAF2 regulates p62 by targeting ubiquitination to exert its cancer-promoting effects.

detailed description

The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. In order to enable those skilled in the art to understand the technical solutions of the present invention more clearly, the technical solutions of the present invention will be described in detail below in conjunction with specific embodiments.

Example 1 Cell lines and culture conditions

Human embryonic kidney epithelial cells 293, human liver cancer cell lines HepG2, Huh7, Hep3B, PLC/PRF/5, SK-HEP-1, human normal liver cells HL-7702 cells, this is a long-term cultured cell line in our laboratory, and the culture conditions : All use DMEM medium (containing 1% penicillin and streptomycin), placed in 5% CO2, 37 ℃ incubator for culture.

Example 2 Real-time PCR detection of the mRNA expression level of TRAF2 in liver cancer cells and normal liver cells

1. RNA extraction

(1) Take the cells cultured in the six-well plate, add 1ml Trizol to lyse the cells, and transfer the liquid to an RNase-free Eppendorf tube.

(2) Add 0.2ml of pre-cooled chloroform, shake vigorously by hand for 15 seconds, and let it stand at room temperature for 3 minutes until clear layering can be seen.

(3) Centrifuge at 12000g for 15 minutes at 4°C, and transfer the upper aqueous phase containing RNA to a new Eppendorf tube.

(4) Add 0.5 ml of pre-cooled isopropanol, mix it upside down and let it stand at room temperature for 10 minutes.

(5) Centrifuge at 10000g for 10 minutes at 4°C; discard the supernatant, and add 1ml 75% ethanol to wash the RNA pellet. Centrifuge at 7500g for 5 minutes at 4°C, and discard the supernatant.

(6) Dry for about 5-10 minutes at room temperature. Add 20μl～50μl of RNase-free water, pipette several times with a pipette tip, and place at 55～60℃ for 10 minutes to completely dissolve the RNA.

(7) Measure the concentration of RNA with NanoDrop instrument.

2. Reverse transcription synthesis of cDNA

Add the following reagents to the RNase-free PCR tube:

Reverse transcription program: 37°C, 15min; 85°C, 5s, end the reaction.

3. Fluorescence quantitative RT-PCR experiment

The primers used in the experiment are derived from the verified primer sequences in published literature, using cDNA as a template, and adding other components one by one according to the kit instructions. The fluorescence quantitative PCR reaction system and procedures are shown in the following table:

PCR program: 95℃ pre-denaturation 30s→95℃5s→60℃30s, repeat 40 cycles;

Make three replicate wells for each sample, and the real-time quantitative PCR instrument will automatically read the Ct value of each sample according to the standard curve to calculate the copy number of each sample, and then take the average of the quantitative results of the two samples. Finally, GAPDH gene was used as the internal reference gene, and the gene copy number of each sample was checked separately.

As shown in Figure 1, through quantitative RT-PCR detection, the expression level of TRAF2mRNA in the 5 hepatocarcinoma cell lines was significantly higher than that of normal HL7702 cells.

Example 3 Detection of TRAF2 protein expression levels in cancer tissues and adjacent tissues of liver cancer patients in clinical specimens.

The present invention has a total of 120 pairs of clinical tissue specimens, among which 90 pairs of tissue chips are used for immunohistochemical staining to detect the expression level of TRAF2, and 30 pairs are used to detect the protein expression level of TRAF2 by Western blot. The tissues are from the First Affiliated Hospital of Zhejiang University.

1. Extraction of tissue protein

(1) Using liquid nitrogen grinding, put the obtained liver cancer clinical sample tissue in a mortar, and add liquid nitrogen for grinding. During the grinding process, ensure that there is always liquid nitrogen in the mortar. When the tissue sample is powdered in the mortar, transfer it to the EP tube and add the protein lysis solution.

(2) Lysis: Protease inhibitor and phosphatase inhibition should be added to the lysis solution in advance, the ratio is: 50:1:5, the lysis is 30min, and the mixing is done every 10min. The entire lysis process is operated on ice. 13600rpm, 4℃, 30min, transfer the supernatant into a new 1.5ml EP tube for later use.

(3) Protein concentration determination: use BCA protein quantification kit for determination, the specific method is in accordance with the kit instructions, and M5 microplate reader is used for reading.

2.Western blot

(1) Preparation of protein gel:

Separating glue:

Concentrated glue:

(2) Preparation of denatured samples: For protein samples whose concentrations have been measured before, add an appropriate amount of 4×SDS sample buffer according to the sample volume, and add water to make up to ensure that the total volume of each sample is the same. 100℃, 10min metal bath, completely denature the protein. Centrifuge at 13600rpm, RT, 1min for later use;

(3) Gel electrophoresis: carefully pull out the gel-making comb, rinse it gently with clean water, fix it in the electrophoresis tank, and add 1×SDS electrophoresis solution; load the prepared protein samples one by one according to the experimental requirements. It should be noted that try to avoid the loss of protein samples during sample loading. The procedure of protein electrophoresis is: 60V→marker clear separation→120V→bromophenol blue to the bottom of the gel, and end the gel run (keep constant pressure).

(4) Transfer membrane: prepare the filter paper, sponge pad, and NC membrane required for transfer membrane in advance, mark them with a pencil and soak them in transfer membrane solution in advance; after electrophoresis, pry off the offset plate, take out the gel and place it on the transfer membrane In the liquid, prepare a "sandwich" transfer membrane structure in the order of sponge pad, filter paper, NC membrane, and protein gel. In this process, air bubbles must be removed at each step, and then translated to the inner tank of the transfer membrane, with the positive electrode underneath. The negative electrode is on top; the film transfer tank is filled with film transfer liquid, and ice-water mixture is added to the periphery to cool down. The film transfer procedure: constant current 250mA, 75min.

(5) Closing: After the transfer of the membrane, 1×TBST cleans the NC membrane once, then the Ponceau staining solution is stained, and the NC membrane is cut according to the position and size of the strip; 1×TBST washes the Ponceau to remove the red, 5% Seal the NC membrane with skimmed milk and proceed slowly on a shaker for 45-60 minutes.

(6) Primary antibody incubation: After blocking, 1×TBST washes the remaining milk, and different target molecules are prepared according to the instructions to prepare the primary antibody diluent. Generally, 1% skimmed milk or 5% BSA is used, placed in a refrigerator at 4°C, and incubated on a shaker. 12-15 hours.

(7) Recover the primary antibody and wash the membrane: After incubating the primary antibody, it is recovered into a 15ml centrifuge tube and placed in a refrigerator at -20°C for storage. Wash the NC membrane 3 times with 1×TBST for 10 minutes each time.

(8) Secondary antibody incubation: According to the properties of the primary antibody, use 3% skimmed milk to configure the corresponding secondary antibody, with a ratio of 1:10000, for 1 hour, at room temperature, and slowly incubate.

(9) Washing the membrane: After the secondary antibody incubation is completed, wash it three times according to the same procedure as washing the primary antibody, and let it stand for WB exposure.

(10) Exposure and image acquisition: ECL 1:1 mixing and color development for 5 minutes, implying that the medium exposure X-ray film, the exposure time is adjusted according to the strength of the target band signal, and then the scanner scans the film, saves the image, and analyzes the results.

As shown in Figure 1, after western blot detection, the protein expression level of TRAF2 in clinical liver cancer tissues was significantly higher than that in adjacent tissues.

Example 4 Immunohistochemical detection of the expression of TRAF2 in liver cancer and adjacent tissues

(1) Baked slices: Place the tissue chip in an oven, adjust the temperature to 63℃, and bake the wax for 1 hour to melt the surface wax

(2) Tissue dewaxing: dewax and hydrate the sections in sequence with xylene for 15 minutes, xylene II for 15 minutes, 100% alcohol for 5 minutes, 100% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, and 75% alcohol for 5 minutes. deal with.

(3) Rinse the sections: Rinse the sections with distilled water three times, each time for 5 minutes; continue to wash three times with PBS, each time for 5 minutes.

(4) Antigen retrieval: use DAKO automatic immunohistochemistry pretreatment system instrument to restore.

(5) Add 3% H2O2 dropwise, incubate at 37°C for 30 minutes, take out the chip, and wash it with PBS three times for 5 minutes each time.

(6) Blocking: Add goat serum blocking solution dropwise, and incubate at 37°C for 30 minutes.

(7) Primary antibody incubation: discard the goat serum blocking solution, dry the surrounding fluid, and directly add the diluted primary antibody dropwise at 4°C overnight.

(8) Rewarming: Take out the wet box and incubate at 37°C for 30 minutes.

(9) Wash with PBS three times, 5 minutes each time. Add the secondary antibody dropwise and incubate at 37°C for 30 min.

(10) Wash three times with PBS, each washing for 5 minutes; add dropwise horseradish peroxidase-labeled streptomycin avidin working solution, and incubate at 37°C for 30 minutes.

(11) Wash with PBS for 5 minutes each time, a total of three times.

(12) DAB color development: DAB color development liquid is configured according to the instructions, DAB color development liquid is added dropwise, the color development time is controlled by observation under a microscope, and the color development is terminated by distilled water.

(13) Hematoxylin counterstaining for 10 minutes.

As shown in Figure 1, the immunohistochemical staining picture shows that the staining intensity of TRAF2 in liver cancer tissues is significantly higher than that in adjacent adjacent tissues. After quantifying the staining intensity, SPSS software was used to analyze the T-test and found that the expression level of TRAF2 in liver cancer tissues was significant Higher than adjacent tissues.

Example 5 ATPlite cell proliferation experiment

(1) Huh7 and Hep3B cells should be pretreated in advance according to the experimental requirements and counted. Inoculate 1500 cells in each well of a 96-well plate, and make three duplicate wells;

(2) On the second day, add 35μl of ATPlite to each well, mix for 5 minutes in the dark, export to a white 96-well plate, and read with a microplate reader;

(3) Repeat the steps for the second day for a total of 5 days of monitoring;

(4) After that, the readings of each day are compared with the first day, and the growth curve is drawn. A total of three replicates were performed for statistical analysis.

As shown in Figure 3, after detection by the ATPlite kit, it was found that after the TRAF2 gene was knocked out in Huh7 and Hep3B cells, the proliferation ability of Huh7 and Hep3B cells was significantly reduced. Figure 4. After the TRAF2 knockout Huh7 cells were re-transformed into the TRAF2 overexpression plasmid, the proliferation ability of Huh7 cells was significantly stronger than that of the control group.

Example 6 Clone formation experiment

Preparation: Add 2.5ml 0.1% gelatin and let it stand for about 20 minutes to solidify the gelatin.

(1) Add 2.5ml 0.1% gelatin to a 60mm petri dish and let it stand for about 20 minutes to solidify the gelatin. Huh7 and Hep3B cell lines were digested, centrifuged and counted. Each 60mm culture dish was inoculated with 300 Huh7 and Hep3B cells, and three replicate holes were made;

(2) Observe the size of the clone under the microscope when the cells grow for about 7-10 days. When the number of cloned cells is more than 50, collect the cells for Coomassie brilliant blue staining;

(3) Discard the culture medium, wash the petri dishes with 1×PBS at room temperature, and aspirate the remaining liquid, add 2.5ml Coomassie dye to each dish, and dye for 20-30 minutes at room temperature;

(4) After dyeing, the dye is recovered, rinsed with clean water, and dried at room temperature;

(5) Using the gel imaging system, select the coomassie mode to take pictures and save, count the number of clones and analyze.

As shown in Figure 3, after testing with the ATPlite kit, it was found that the proliferation ability of Huh7 and Hep3B cells was significantly reduced after TRAF2 was knocked out.

As shown in Figure 3, after Coomassie brilliant blue staining, it was found that after the TRAF2 gene was knocked out in Huh7 and Hep3B cells respectively, the clone formation of Huh7 and Hep3B cells decreased significantly. Figure 4. After the TRAF2 knockout Huh7 cells were re-transformed into the TRAF2 overexpression plasmid, the Huh7 cells clone formation ability was significantly stronger than the control group.

Example 7 Subcutaneous tumor formation experiment in nude mice

(1) Cell preparation: Huh7 cells with TRAF2 knocked out and control cells are cultured normally, and cells with good growth status are selected for passage counting;

(2) Preparation of nude mice: 4-5 weeks old, immunodeficient male nude mice were purchased from Jiangsu Jicui Yaokang Biotech Co., Ltd. The nude mice were raised in a room dedicated to immunodeficiency mice, and each nude mouse was marked with ear studs and weighed. weight. According to the experimental design, there are 5 nude mice per group;

(3) Cell count: The number of cells inoculated is 1×10 ⁶ cells, the cells are digested and centrifuged and counted, and the cells are resuspended in 1×PBS to make the concentration of the cell suspension 1×10 ⁷ cells/ml. After fully mixing the cell suspension, each group is divided into 6 EP tubes, each tube 100μl;

(4) Cell inoculation: inoculation method: subcutaneous inoculation; inoculation site: the position of the side abdomen of nude mice near the buttocks; before inoculation, rinse the insulin needle with one tube of cell suspension and inoculate 100 μl of the cell suspension;

(5) Tumor volume measurement and nude mouse weighing: Weigh the nude mouse body weight and tumor length and short diameter every three days and record;

(6) When the maximum diameter of the tumor grew to about 1-1.5cm, the nude mice were sacrificed by cervical dislocation, the subcutaneous tumor was taken out and photographed, weighed, and part of the tumor tissue was divided for WB, part for RT-PCR, and part for In immunohistochemistry (fixed in tissue specimen fixative), the remaining specimens are frozen in liquid nitrogen for later use.

As shown in Figure 4, Huh7-sgTRAF2 cells and Huh7-sgCtrl cells were prepared into cell suspensions and inoculated into nude mice subcutaneously. After 14 days of observation and recording, it was found that the volume and weight of subcutaneous tumors formed after TRAF2 were knocked out were less than Control group.

Example 8 Co-immunoprecipitation experiment (Co-IP)

1. External Co-IP

(1) The cells are plated the day before, and the cells are transfected with plasmid on the second day (the specific amount of plasmid is based on the experimental design). After 48 hours of transfection, the cells are scraped to collect the cells. You can proceed directly to the second step or put the cells at -80°C for use;

(2) Lyse the cells, collect the cell supernatant and transfer it to the EP tube for later use. Reserve 45μl of whole cell lysate and add 15μl 4×SDS sample buffer as a control (WCE). If the interaction is quantitatively detected, the protein concentration is required; if it is only qualitative, proceed directly to the third step of the experiment;

(3) Take an appropriate amount of antibody-conjugated beads, add 1ml of lysis buffer without protease inhibitor to wash off the beads protection solution, centrifuge at 4°C, 5000rpm for 1min, and discard the supernatant as much as possible.

(4) Transfer the remaining supernatant to the prepared beads, and incubate for 3 hours in a refrigerator at 4°C on a vertical shaker;

(5) Beads eluted, centrifuge at 5000 rpm at 4°C for 1 min, discard the supernatant, and centrifuge at 5000 rpm for 30 seconds to aspirate the remaining liquid. Add 1ml wash buffer to the beads, centrifuge at 5000rpm for 1min at 4℃ to wash the beads, repeat 4 times. After the last centrifugation, centrifuge for another 30s, and suck up the remaining liquid with a pipette tip;

(6) Add an appropriate amount of 2×SDS sample buffer to the beads, 1100rpm, shake for 15s, repeat twice;

(7) Metal bath with WCE, 100℃, WCE 10min, IP sample 5min;

(8) After protein denaturation, centrifuge at 13600 rpm for 1 min at RT, and then perform WB detection.

2. Endogenous Co-IP

The endogenous co-immunoprecipitation step is similar to the exogenous one, with an additional step of incubating antibodies: add specific antibodies and Normal IgG as controls to the cell lysis supernatant, and incubate in a refrigerator at 4°C for 2-4 hours; prepare 20μl Protein G beads , The method is the same as that of exogenous. Transfer all the samples that have been incubated with antibodies into EP tubes with added Protein G beads, and continue to incubate in a refrigerator at 4°C for 3 hours; as shown in Figure 5, after exogenous and endogenous Co- IP detection found that TRAF2 binds to p62.

Example 9 Detection of Ubiquitination Level

In this experiment, the p62 protein ubiquitination level was detected by the exogenous Co-IP method. Since the plasmid with Ub in the experiment is HA tag, the experiment was carried out with HA beads. Collect the samples, lyse the protein and determine the concentration, add 15μl HA beads to each group of samples, and then co-incubate with the beads for subsequent steps. As shown in Figure 6, the ubiquitin E3 ligase TRAF2 can add ubiquitin chains to p62.

Embodiment 10 Data analysis

In the present invention, the survival analysis adopts the Kaplan-Meier method to analyze, and the experimental data adopts SPSS 20.0 software for statistical analysis. Analysis of variance (ANOVA) and Student’s t-test test were used to test the differences between the cell proliferation and survival experiment groups. P<0.05 indicates that the differences are statistically significant. The results are graphed by GraphPad Prism 8.0 software.

The description of the above-mentioned embodiments is only for understanding the method of the present invention and its core idea. It should be pointed out that the implementation of the present invention is not limited by the above-mentioned examples. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention. These improvements and Modifications will also fall within the protection scope of the claims of the present invention.

Claims (9)

1. A marker for diagnosing liver cancer, and the marker is TRAF2.
2. The use of the marker of claim 1 in the preparation of reagents or kits for screening diagnosis and treatment of liver cancer.
3. The use of the marker of claim 1 in the preparation of a medicine or a pharmaceutical composition for preventing or treating liver cancer, the pharmaceutical composition comprising a TRAF2 functional expression inhibitor.
4. Use of the marker of claim 1 in screening drugs for diagnosis and treatment of liver cancer.
5. A reagent for detecting the expression level of the marker of claim 1, the reagent comprising the following primers:

   TRAF2-F: 5'-GCCCTTCAACCAGAAGGTGAC-3’

   TRAF2-R: 5'-CCAACCCCCAGACACCAGTA-3'.
6. The reagent according to claim 3, wherein the amplification method of the amplification primer is TaqMan probe method.
7. The application of the reagent according to claim 2 in diagnosing liver cancer and predicting metastasis of liver cancer.
8. A kit for diagnosing liver cancer, comprising a reagent for detecting the marker according to claim 1.
9. The use of the kit of claim 6 in diagnosing liver cancer and predicting liver cancer metastasis.

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