Hepatocellular carcinoma protein marker and application thereof

Abstract

The invention provides a hepatocellular carcinoma protein marker and application thereof, and application of KCNJ12 as a molecular marker of hepatocellular carcinoma in preparation of products for detection, prognosis or treatment of hepatocellular carcinoma. The molecular marker is a KCNJ12 gene or protein; the hepatocellular carcinoma protein marker KCNJ12 provided by the invention can be used as a target for developing drugs for preventing and treating HCC, and has important potential application value.

Description

Technical Field

[0001] The present invention relates to a protein marker for hepatocellular carcinoma and its application, belonging to the technical field of tumor molecular biology. Background Art

[0002] The incidence and mortality of primary liver cancer are both relatively high. Primary liver cancer mainly includes hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular carcinoma-cholangiocarcinoma, etc. Among them, HCC is the most common primary liver cancer, accounting for about 75-85% of primary liver cancer. The main risk factors for HCC include long-term alcohol consumption, diabetes or obesity-related non-alcoholic fatty liver (NASH), hepatitis B virus and hepatitis C virus infections.

[0003] Currently, the diagnostic methods for liver cancer mainly include tumor marker detection, imaging examination, pathological biopsy, etc. However, when patients are diagnosed, they often have reached the advanced stage of cancer. Therefore, it is urgent to study the potential molecular mechanisms of the occurrence and development of liver cancer for the diagnosis and treatment of liver cancer.

[0004] Tumor marker detection is a method that can screen tumor patients earlier among many diagnostic methods for liver cancer. In the prior art, the tumor markers for liver cancer include alpha-fetoprotein, CA199, as well as fucosidase, carcinoembryonic antigen, and ferritin, and also include abnormal prothrombin and gamma-glutamyl transferase isoenzyme, etc. Among them, alpha-fetoprotein is a relatively specific tumor marker, which can be used for the definite diagnosis and early diagnosis of liver cancer, and can also be used to judge the treatment effect and recurrence signs. For patients with significantly elevated alpha-fetoprotein, the possibility of liver cancer needs to be considered in combination with the medical history.

[0005] In addition, experimental cancer cell lines include low-metastasis cell lines, high-metastasis cell lines, low-invasive cell lines, and high-invasive cell lines. According to different experimental design requirements, the required cancer cell lines can be selectively screened. Therefore, different types of cancer cell lines are also the needs of scientific experiments, and different types of cancer cell lines, as commodities, also have broad economic value. Summary of the Invention

[0006] The present invention provides that the protein marker KCNJ12 for hepatocellular carcinoma can be used as a target for developing drugs for preventing and treating HCC, and has important potential application value.

[0007] To achieve this purpose, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, there is provided the use of KCNJ12 as a molecular marker for hepatocellular carcinoma in the preparation of products for the detection, prognosis or treatment of hepatocellular carcinoma, characterized in that the molecular marker is the KCNJ12 gene or protein.

[0009] Preferably, the expression level of KCNJ12 is significantly up-regulated in the hepatocellular carcinoma tissue or cells of hepatocellular carcinoma patients as compared with normal healthy human liver tissues or cells or the adjacent tissues or cells of hepatocellular carcinoma patients.

[0010] In the present invention, the overall survival rate of HCC patients with high expression of KCNJ12 is significantly lower than that of the low-expression KCNJ12 group, indicating that the prognosis of patients with high expression of KCNJ12 is poor. It can be seen that KCNJ12 is highly expressed in HCC, and high expression is associated with poor prognosis.

[0011] Preferably, the KCNJ12 can promote the proliferation, self-renewal ability, migration and invasion ability of hepatocellular carcinoma cells.

[0012] In the present invention, through in vitro experiments of knocking out KCNJ12, it is found that knocking out KCNJ12 can significantly inhibit the proliferation, self-renewal ability, migration and invasion ability of HCC cells.

[0013] Preferably, the hepatocellular carcinoma cell lines include PLC, HepG2, and PVTT.

[0014] In the second aspect of the present invention, there is provided the use of a KCNJ12 inhibitor in the preparation of a pharmaceutical composition for preventing or treating hepatocellular carcinoma.

[0015] Preferably, the KCNJ12 inhibitor is siRNA, dsRNA, shRNA, miRNA, antisense nucleotides that can reduce the expression level of KCNJ12; or constructs that can express or form the siRNA, dsRNA, shRNA, miRNA, antisense nucleotides.

[0016] Preferably, the hepatocellular carcinoma cell lines include PLC, HepG2, and PVTT.

[0017] In the third aspect of the present invention, there is provided the use of a KCNJ12 protein detection reagent in the preparation of a reagent for evaluating the prognosis of hepatocellular carcinoma.

[0018] In the present invention, the KCNJ12 protein detection reagent can be an ELISA kit.

[0019] Preferably, the hepatocellular carcinoma cell lines include PLC, HepG2, and PVTT.

[0020] The fourth aspect of the present invention provides a method for constructing a low-migration and low-invasiveness hepatocellular carcinoma cell line for experimental use, which is characterized by including the following steps:

[0021] S1. Construction of KCNJ12 knockout virus;

[0022] S2. Infecting the constructed virus into hepatocellular carcinoma cells;

[0023] S3. Culturing to obtain a low-migration and low-invasiveness hepatocellular carcinoma cell line.

[0024] Preferably, the hepatocellular carcinoma cell line includes PLC, HepG2, and PVTT.

[0025] In the present invention, the low-migration and low-invasiveness hepatocellular carcinoma cell line for experimental use can be used in experiments such as tumor research and tumor drug research. It has good commercial value.

[0026] The fifth aspect of the present invention provides a kit for detecting or prognosticating hepatocellular carcinoma, including a reagent for detecting KCNJ12 protein.

[0027] In the present invention, the reagent for detecting KCNJ12 protein can be an ELISA kit.

[0028] Preferably, the hepatocellular carcinoma cell line includes PLC, HepG2, and PVTT.

[0029] Compared with the prior art, the beneficial effects and remarkable progress of applying the technical solution of the present invention are as follows:

[0030] 1. The present invention provides a HCC biomarker KCNJ12, which can not only be used as a biomarker for HCC diagnosis or / and prognostic evaluation, but also can be used as a target for developing drugs for preventing and treating HCC, and has important potential application value;

[0031] 2. The present invention verifies that the overall survival rate of HCC patients with high expression of KCNJ12 is significantly lower than that of the low-expression KCNJ12 group, indicating that the prognosis of high-expression KCNJ12 is poor. It can be seen that KCNJ12 is highly expressed in HCC, and high expression is related to poor prognosis;

[0032] 3. Through in vitro experiments of knocking out KCNJ12, the present invention finds that knocking out KCNJ12 can significantly inhibit the proliferation, self-renewal ability, migration and invasion ability of HCC cells. It can be seen that KCNJ12 can also be used for preventing and treating HCC, can be used as a target for preparing drugs for preventing and treating HCC, and can prepare a low-migration and low-invasiveness hepatocellular carcinoma cell line. Description of the Drawings

[0033] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0034] Figure 1 It is the poor prognosis of KCNJ12 in Example 1 in the population with high mutation load;

[0035] Figure 2 It is the effect of detecting the knockout of KCNJ12 on cell proliferation by the CCK8 method in Example 2;

[0036] Figure 3 It is the result of the colony formation experiment in Example 3;

[0037] Figure 4 It is the effect of knocking out KCNJ12 on cell migration in Example 4;

[0038] Figure 5 It is the effect of knocking down KCNJ12 on cell invasion in Example 5. Detailed implementation manners

[0039] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further elaborated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions indicated in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0040] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners of this application.

[0041] To more fully understand the present invention, the following explains the professional terms in the present invention.

[0042] KCNJ12, potassium inwardly rectifying channel subfamily J member 12 (KCNJ12), is located on chromosome 17p11.1 and encodes a 48 kDa protein.

[0043] PLC, PLC / PRF / 5 human hepatoma Alexander cells, this cell line secretes hepatitis B virus surface antigen (HBsAg). This cell line was originally contaminated with mycoplasma and the mycoplasma was removed with BM-cycline.

[0044] HepG2 cells are derived from the liver cancer tissue of a 15-year-old Caucasian. These cells secrete various plasma proteins, such as albumin, α2-macroglobulin, plasminogen, transferrin, etc. These cells express 3-hydroxy-3-methylglutaryl coenzyme A reductase and hepatic triglyceride lipase. The mRNA expression of catalase increases and the mRNA expression of ApoA-I decreases in the presence of paraquat in these cells.

[0045] PVTT cells, portal vein tumor thrombus. Liver cancer patients often have portal vein tumor thrombus.

[0046] Tumor refers to a new growth (neogrowth) formed by the hyperplasia of local tissue cells under the action of various tumorigenic factors in the body. Because this new growth often appears as a mass-like protrusion occupying space, it is also called a neoplasm. Liver cancer and pancreatic cancer are both known as the "kings of cancer" and are tumors with extremely high malignancy. Research has found that tumor cells will show metabolic changes different from normal cells, and at the same time, tumor cells themselves can adapt to changes in the metabolic environment through the conversion between glycolysis and oxidative phosphorylation (OXPHOS).

[0047] A kit refers to a box used to hold chemical reagents for detecting chemical components, drug residues, or virus types, etc. It is generally used in hospitals, inspection and quarantine, or pharmaceutical enterprises. A kit is an important tool in chemical analysis experiments, and its function is to detect and analyze the substances contained in the sample. In biochemical detection, a kit is also an important method for discovering diseases. Kits can be used in detection methods such as enzyme-linked immunosorbent assay and immunoassay.

[0048] Protein expression refers to a molecular biology technique that uses model organisms such as bacteria, yeast, animal cells, or plant cells to express foreign gene proteins. It occupies a core position in genetic engineering technology.

[0049] Antisense nucleotides are a class of molecular sequences that specifically bind to target gene DNA or mRNA through sequences to inhibit the expression of this gene and regulate at the gene level.

[0050] Example 1 Relationship between KCNJ12 and the prognosis of HCC patients

[0051] The purpose of this example is to count the overall survival rates of HCC patients in the high-expression KCNJ12 group and the low-expression KCNJ12 group to compare the relationship between KCNJ12 and the prognosis of HCC.

[0052] The results are as Figure 1As shown, KCNJ12 shows a poor prognosis in the high mutation burden population. The overall survival rate of HCC patients with high expression of KCNJ12 is significantly lower than that of the low expression KCNJ12 group, indicating that the prognosis of high expression of KCNJ12 is poor. The above results suggest that KCNJ12 is highly expressed in HCC, and high expression is associated with poor prognosis.

[0053] Example 2 Relationship between KCNJ12 and cancer cell proliferation ability

[0054] 2.1. The experimental cells selected were human hepatocellular carcinoma cells (HCC): PLC, HepG2, and PVTT cell lines;

[0055] 2.2. Compare the cell proliferation ability of PLC, HepG2, and PVTT, three kinds of cells, with or without knocking out KCNJ12. Among them, the CCK8 kit (Absin, abs50003-50ml) was used to detect the cell proliferation ability. The specific detection steps were as follows: When the three HCC cells were in the logarithmic growth phase, the cells were digested and resuspended with 10% FBS + 1% PS high-glucose DMEM. The cells were counted at 1000 cells per well (100uL) and inoculated into 96-well plates (cultured with 10% FBS + 1% PS high-glucose DMEM medium). Each experimental condition was set with 6 replicates. 10ul of CCK8 reagent was added to each well, and the cells were incubated at 37°C for 2h. The absorbance value of the samples was detected at 450nm by an enzyme-linked immunosorbent assay (model), and the samples were continuously detected and recorded for 5 days.

[0056] The results are as Figure 2 shown. In the figure, A is the growth curve of PLC cells after knocking out KCNJ12 detected by the CCK8 method; B is the growth curve of HepG2 cells after knocking out KCNJ12 detected by the CCK8 method; C is the results of the scratch experiment of PVTT cells at 24h and 48h after knocking out KCNJ12. The figure was drawn by GraphPad prism 8.0 (*p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001). The above results indicate that the cell proliferation ability of the three cancer cells decreased significantly after knocking out KCNJ12.

[0057] Example 3 Colony formation assay

[0058] 3.1. The experimental cells selected were human hepatocellular carcinoma cells (HCC): PLC, HepG2, and PVTT cell lines;

[0059] 3.2. When the cells grow to the logarithmic growth phase, digest them and resuspend the cells with high-glucose DMEM containing 10% FBS + 1% PS. Uniformly seed the HCC cells in a six-well plate, with 1500 cells per well (total volume 2 ml), three parallels in each group. Incubate them at 37°C in a 5% CO2 cell culture incubator with high-glucose DMEM medium containing 10% FBS + 1% PS for 11 - 14 days. During this period, change the medium every 3 days. When visible monoclonal colonies appear, aspirate the upper cell culture medium, wash the cell surface once with ice-cold 1×PBS, add 1 ml of 4% paraformaldehyde (Shanghai Ruiyu Biotechnology Co., Ltd., #Bry-0019-5) and fix at room temperature for 15 min, stain with 0.1% crystal violet (product number) at room temperature for 15 min, wash the residual staining solution on the cell surface, invert the culture plate and let it dry, then take pictures.

[0060] The results are as Figure 3 shown. A shows the ability of PLC cells to form clones after knocking out KCNJ12; the ability of HepG2 cells to form colonies after knocking out KCNJ12; the ability of PVTT cells to form colonies after knocking out KCNJ12; B shows the quantitative graph of the data 14 days after PLC cells form clones after knocking out KCNJ12; the quantitative graph of the data 14 days after HepG2 cells form clones after knocking out KCNJ12; the quantitative graph of the data 14 days after PVTT cells form clones after knocking out KCNJ12. The figure was drawn by GraphPad prism 8.0 (*p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001). The above results indicate that the number of cancer cell clones significantly decreases after knocking out KCNJ12 in the three types of cancer cells.

[0061] Example 4. Effect of knocking out KCNJ12 on the migration of cancer cells

[0062] Use the transwell assay to detect the migration ability of HCC cells (PLC, HepG2, PVTT cell lines), and select a chamber with a pore size of 8 μm (greiner bio-one, 662638) and 24-well transwell plates (greiner bio-one, 662160).

[0063] When the cells are cultured to the logarithmic growth phase, digest the cells, wash the cells once with ice-cold 1X PBS, resuspend the cells with FBS-free high-glucose medium, count and plate the cells, and set 3 replicate wells for each group condition. Add 5×10^4 cells (200 μl FBS-free high-glucose medium) to the upper chamber, and add 500 μl complete medium (10% FBS + 1% PS + high-glucose DMEM) to the lower chamber. After culturing in a 37°C incubator for 24 h, use a cotton swab to wipe off the cells that have not passed through the upper chamber, fix the lower surface of the chamber with 4% paraformaldehyde at room temperature for 15 min, stain with 0.1% crystal violet at room temperature for 15 min, wash the residual staining solution on the cell surface, invert and air-dry, and then examine under a microscope and take pictures for statistics.

[0064] The results are as Figure 4 shown. A shows the results of the cell migration assay after knocking out KCNJ12 in PLC cells; the results of the cell migration assay after knocking out KCNJ12 in HepG2 cells; the results of the cell migration assay after knocking out KCNJ12 in PVTT cells; B shows the data quantification graph of cell migration 72 h after knocking out KCNJ12 in PLC cells; the data quantification graph of cell migration 72 h after HepG2 cells; the data quantification graph of cell migration 48 h after knocking out KCNJ12 in PVTT cells. The figure was drawn by GraphPad prism 8.0 (*p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001). The above results indicate that the migration ability of the three types of cancer cells decreases after knocking out KCNJ12.

[0065] Example 5 Effect of knocking out KCNJ12 on cell invasion

[0066] Use the transwell assay to detect the invasion ability of HCC cells (PLC, HepG2, PVTT cell lines), and select a chamber with a pore size of 8 μm (greiner bio-one, 662638) and 24-well transwell plates (greiner bio-one, 662160).

[0067] Thaw Matrigel on ice (Corning, 356231), dilute Matrigel with ice-cold high-glucose DMEM medium without FBS (gel: FBS-free medium = 1:11), aspirate 100 μl of the diluted Matrigel and add it to the upper chamber, and activate it in an incubator at 37°C for 2 h. Digest the cells, wash the cells once with ice-cold 1X PBS, resuspend the cells with high-glucose DMEM medium without FBS, count the cells and plate them, and set up 3 replicate wells for each condition. Add 5×10^4 cells (200 μl of high-glucose DMEM medium without FBS) to the upper chamber, and add 500 μl of high-glucose DMEM medium containing 10% FBS + 1% PS to the lower chamber. After culturing in an incubator at 37°C for 48 h, use a cotton swab to wipe off the cells in the upper chamber that did not pass through the membrane, fix the lower surface of the chamber with 4% paraformaldehyde at room temperature for 15 min, stain with crystal violet at room temperature for 15 min, wash the residual staining solution on the cell surface, invert and air-dry, and examine under a microscope and take pictures for statistics.

[0068] The results are as Figure 5 shown. A shows the results of the cell invasion experiment after knocking out KCNJ12 in PLC cells; the results of the cell invasion experiment after knocking out KCNJ12 in HepG2 cells; the results of the cell invasion experiment after knocking out KCNJ12 in PVTT cells; B shows the data quantification graph of the cell invasion after 48 h in PLC cells after knocking out KCNJ12; the data quantification graph of the cell invasion after 48 h in HepG2 cells; the data quantification graph of the cell invasion after 48 h in PVTT cells after knocking out KCNJ12. The figure was drawn by GraphPad prism 8.0 (*p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001). The above results indicate that the invasive ability of the three types of cancer cells decreases after knocking out KCNJ12.

[0069] The applicant declares that in the process of the above description in the specification:

[0070] The descriptions of terms such as "this embodiment", "the embodiment of the present invention", "as shown in...", "further", "further improved technical solution", etc. mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example, and moreover, the specific features, structures, materials or characteristics described can be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, on the premise of not generating contradictions, those of ordinary skill in the art can combine or combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0071] Finally, it should be noted that:

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it;

[0073] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features. These modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art according to the content of this specification all fall within the scope claimed by the present invention.

Claims

1. Use of KCNJ12 as a molecular marker for hepatocellular carcinoma in the preparation of a product for detection, prognosis or treatment of hepatocellular carcinoma, characterized in that: The molecular marker is KCNJ12 gene or protein.

2. The use according to claim 1, characterized in that Compared with normal healthy human liver tissue or cells or adjacent cancer tissue or cells of hepatocellular carcinoma patients, the expression level of KCNJ12 in hepatocellular carcinoma tissue or cells of hepatocellular carcinoma patients was significantly upregulated.

3. The use according to claim 1, characterized in that The KCNJ12 can promote the proliferation, self-renewal, migration and invasion ability of hepatocellular carcinoma cells.

4. The use according to claim 3, characterized in that The hepatocellular carcinoma cell lines include PLC, HepG2, and PVTT.

5. Use of KCNJ12 inhibitors in the preparation of pharmaceutical compositions for preventing or treating hepatocellular carcinoma.

6. The use according to claim 5, characterized in that The KCNJ12 inhibitor is an sgRNA, siRNA, dsRNA, shRNA, miRNA, or antisense nucleotide that can reduce the expression level of KCNJ12; or a construct that can express or form the sgRNA, siRNA, dsRNA, shRNA, miRNA, or antisense nucleotide.

7. The use according to claim 3, characterized in that The hepatocellular carcinoma cell lines include PLC, HepG2, and PVTT.

8. Application of KCNJ12 protein detection reagent in the preparation of reagents for evaluating the prognosis of hepatocellular carcinoma.

9. A method for constructing a liver cancer cell line with low migration and low invasion for experimental use, characterized in that: The following steps are involved: S1, Construction of KCNJ12 knockout virus; S2, infecting liver cancer cells with the constructed virus; S3. Cultivate and obtain liver cancer cell lines with low migration and invasion ability.

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