Application of DHCR24 protein acetylation in the treatment and diagnosis of liver cancer

Abstract

The present invention relates to the application of DHCR24 protein acetylation in the treatment and diagnosis of liver cancer, and belongs to the field of biomedical technology. In order to solve the problem of the current lack of efficient clinical treatment drugs and early screening methods for liver cancer, the present invention provides the application of DHCR24 protein acetylation in the treatment and diagnosis of liver cancer, including the application of the acetylation level of the K254 site of the DHCR24 protein as a target for liver cancer treatment in the preparation of drugs for preventing and treating liver cancer, and the application of the acetylation level of the K254 site of the DHCR24 protein as a diagnostic marker in the preparation of a kit for liver cancer diagnosis and prognosis evaluation. The present invention confirms that the increase of acetylation of the DHCR24 protein at the K254 site stabilizes the protein and promotes its enzyme activity and carcinogenicity in HCC cell lines. Therefore, targeted inhibition of the acetylation modification level of the K254 site of the DHCR24 gene is an effective and feasible new strategy for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of DHCR24 protein acetylation in the treatment and diagnosis of liver cancer. Background Art

[0002] Liver cancer, especially primary hepatocellular carcinoma (HCC), is a global public health problem. According to statistics, there were 410,000 new liver cancer patients in my country in 2020, accounting for 9% of the total new liver cancer patients and 13% of the total number of cancer deaths in my country in 2020. The five-year survival rate is only 11.7%-14.1%, which is the second leading cause of cancer death in China and seriously affects the life and health of the Chinese people. The main reason is that early liver cancer is not easy to detect, and it is already in the middle and late stages when it is diagnosed. In addition, the clinical medication effect of late-stage liver cancer is poor and is accompanied by severe drug resistance and high recurrence rate. The tumor volume of early liver cancer is small, and the commonly used liver cancer screening methods such as abdominal ultrasound and CT often cannot detect liver cancer early. At present, there is still a lack of efficient clinical treatment drugs and early screening methods for liver cancer. Summary of the invention

[0003] In order to solve the problem of lack of efficient clinical treatment drugs and early screening methods for liver cancer, the present invention provides the application of DHCR24 protein acetylation in the treatment and diagnosis of liver cancer.

[0004] The technical solution of the present invention:

[0005] Application of the acetylation level of 24-dehydrocholesterol reductase DHCR24 protein K254 site as a target for liver cancer treatment in the preparation of drugs for preventing and treating liver cancer.

[0006] Furthermore, the drug for preventing and treating liver cancer contains irbesartan as the only active ingredient or one of the active ingredients.

[0007] Furthermore, the irbesartan inhibits liver cancer by inhibiting DHCR24 gene expression and acetylation.

[0008] Furthermore, the drug for preventing and treating liver cancer is administered orally, and the daily oral dose of irbesartan is 60 to 100 mg / kg body weight.

[0009] Furthermore, the dosage form of the drug for preventing and treating liver cancer is granules, tablets, capsules, pills or oral liquid preparations.

[0010] Application of the acetylation level of 24-dehydrocholesterol reductase DHCR24 protein K254 site as a diagnostic marker in the preparation of a kit for diagnosis and prognosis assessment of liver cancer.

[0011] A liver cancer diagnosis kit comprises a reagent for detecting the acetylation level of K254 site of DHCR24 protein.

[0012] Furthermore, the reagent for detecting the acetylation level of the K254 site of the DHCR24 protein is a specific antibody, a polypeptide or a small molecule preparation.

[0013] Furthermore, the specific antibody is an acetylated polyclonal antibody of the K254 site of the DHCR24 protein, the polyclonal antibody is prepared by animal immunization and antibody separation and purification using the acetylated peptide-KLH as an antigen, and the amino acid sequence of the acetylated peptide is AK(ace)FTHESQRQEN+Cys.

[0014] Furthermore, the preparation method of the acetylated polyclonal antibody of the DHCR24 protein K254 site is as follows:

[0015] The acetylated peptide-KLH antigen was immunized in New Zealand white rabbits, with conventional immunization for 4 times and additional immunization for 2 times, and then the indirect ELISA method was used to detect the antibody titer after the 3rd, 4th, 5th and 6th immunizations with the screening peptide as the antigen; when the antibody titer exceeded 1:32000, the rabbit serum was collected and the antibody was purified using antigen-specific affinity; the amino acid sequence of the screening peptide was AKFTHESQRQEN+Cys.

[0016] Beneficial effects of the present invention:

[0017] The present invention takes the discovery of the increased acetylation modification level of the K254 site of the 24-dehydrocholesterol reductase DHCR24 protein in liver cancer patients as the starting point, and confirms in clinical samples that the acetylation level of the K254 site of the DHCR24 protein is associated with a relatively poor prognosis of liver cancer. Further, stable cell lines overexpressing different DHCR24 mutant plasmids were generated through in vitro experiments to simulate acetylation (KQ mutation) or deacetylation (KR mutation) in DHCR24 cells, confirming that the increase in acetylation of the DHCR24 protein at the K254 site stabilizes the protein and promotes its enzymatic activity and carcinogenicity in HCC cell lines.

[0018] In nude mouse transplant tumors and AAV8-induced transgenic mouse models, compared with the transplant vector group (Vector), simulated acetylation (K254Q) and overexpressed DHCR24-WT had similar tumorigenic effects, and acetylation loss (K254R) weakened the oncogenic function of DHCR24, indicating that increased acetylation levels at the K254 site of the DHCR24 protein are necessary for the tumorigenic activity of DHCR24 in HCC. Further, in HCC cell models and Den combined with CCL4-induced liver cancer mouse models, it was found that irbesartan inhibited the occurrence and development of liver cancer by targeting the inhibition of DHCR24 gene expression and acetylation levels, which confirmed that targeted inhibition of the acetylation modification level at the K254 site of the DHCR24 gene is an effective and feasible new strategy for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison of the expression levels of DHCR24 protein in paracancerous tissues and liver cancer tissues in two different liver cancer clinical cohorts in Example 1, A is the liver cancer clinical cohort of the Fourth Affiliated Hospital of Harbin Medical University, and B is the liver cancer clinical cohort of Fudan University;

[0020] Figure 2 This is a comparison result of the Western blot test in Example 1 to detect the expression level of DHCR24 gene protein in paracancerous tissue and liver cancer tissue, N represents paracancerous tissue, and T represents liver cancer tissue;

[0021] Figure 3 This is the IHC color image of the gene chip at different DHCR24 protein expression levels in Example 1;

[0022] Figure 4 is a graph showing the relationship between the expression level of DHCR24 protein and the survival rate of liver cancer patients in Example 1;

[0023] Figure 5 A comparison chart of acetylated proteomics sequencing results of HCC samples and non-tumor control samples in Example 2;

[0024] Figure 6 This is a comparison result of Western blot test in Example 2 to detect the acetylation level of K254 site of DHCR24 gene protein in paracancerous tissue and liver cancer tissue, N represents paracancerous tissue, and T represents liver cancer tissue;

[0025] Figure 7 This is the IHC color image of the gene chip of different acetylation levels of DHCR24 protein K254 site in Example 2;

[0026] Figure 8 is a graph showing the relationship between the acetylation level of the K254 site of the DHCR24 protein and the survival rate of liver cancer patients in Example 2;

[0027] Fig. 9 WB images of different MHCC97H and SKHep1 cell models constructed in Example 3;

[0028] Fig.10 From left to right are the cell index comparison diagrams of different MHCC97H and SKHep1 cell models in Example 3;

[0029] Fig.11 These are photos of cell clones formed by different MHCC97H and SKHep1 cell models in Example 3;

[0030] Fig.12 From left to right are comparison diagrams of cell clone formation of different MHCC97H and SKHep1 cell models in Example 3;

[0031] Fig.13 Schematic diagram and WB image of the construction method of hepatocyte DHCR24-specific knockout mice in Example 3;

[0032] Fig.14 Schematic diagram of the modeling process of liver cancer models of wild-type mice and Dhcr24 hepKO mice in Example 3;

[0033] Fig.15 The liver tissue photos and H&E staining microphotographs of wild-type and Dhcr24 hepKO liver cancer mice in Example 3;

[0034] Fig.16 It is a comparison chart of tumor incidence, tumor number and tumor burden of wild-type and Dhcr24 hepKO liver cancer mice in Example 3;

[0035] Fig.17 This is a comparison of blood AFP, ALT and AST levels in wild-type and Dhcr24 hepKO liver cancer mice in Example 3;

[0036] Fig.18 This is a comparison of the body weights of wild-type and Dhcr24 hepKO liver cancer mice in Example 3;

[0037] Fig.19 WB images of stable cell lines overexpressing different DHCR24 mutant plasmids generated in Example 4;

[0038] Fig. 20 This is a WB image of DHCR24 protein expression in different DHCR24 mutant cells after CQ treatment in Example 4;

[0039] Fig.21 and Fig. 22They are respectively WB images of DHCR24 protein expression in MHCC97H-sgDHCR24 cells with different mutations after CHX treatment in Example 4 and a comparison of the relative amounts of DHCR24 protein;

[0040] Fig.23 and Fig.24 They are respectively WB images of DHCR24 protein expression in SKHep1-sgDHCR24 cells with different mutations after CHX treatment in Example 4 and a comparison of the relative amounts of DHCR24 protein;

[0041] Fig.25 This is a comparison of intracellular cholesterol levels in stable cell lines overexpressing different DHCR24 mutant plasmids in Example 4;

[0042] Fig.26 From left to right are the cell index comparison diagrams of the stable cell lines MHCC97H and SKHep1 overexpressing different DHCR24 mutant plasmids in Example 4;

[0043] Fig. 27 and Fig.28 They are respectively a photograph of cell clones formed by stable cell lines overexpressing different DHCR24 mutant plasmids in Example 4 and a comparison diagram of cell clone formation;

[0044] Fig.29 This is a comparison of the actual tumors in nude mice after transplantation of stable cell lines expressing different DHCR24 variants in Example 5;

[0045] Fig.30 and Fig.31 They are comparative diagrams of the weight and volume of tumors in nude mice after transplantation of stable cell lines expressing different DHCR24 variants in Example 5;

[0046] Fig.32 This is a comparison of the overall survival of nude mice after transplantation of stable cell lines expressing different DHCR24 variants in Example 5;

[0047] Fig.33 Schematic diagram of the modeling process of establishing DHCR24 heavy-expressing mouse Dhcr24 hepKO by controlling Alb-Cre and AAV in Example 5;

[0048] Fig.34 This is a comparison chart of tumor incidence and tumor number in the control Alb-Cre and different DHCR24 heavy-expressing mice Dhcr24 hepKO in Example 5;

[0049] Fig.35 This is a comparison of serum AFP, ALT, and AST levels in the control Alb-Cre and different DHCR24 heavy-expressing mice Dhcr24 hepKO in Example 5;

[0050] Fig.36 This is a comparison of serum cholesterol and liver cholesterol levels in the control Alb-Cre and different DHCR24 heavy-expressing mice Dhcr24 hepKO in Example 5;

[0051] Fig.37 This is a comparison of serum 7-KC and liver 7-KC levels in the control Alb-Cre and different DHCR24 heavy-expressing mice Dhcr24 hepKO in Example 5;

[0052] Fig.38 The liver tissue photos and H&E staining microphotographs of the control Alb-Cre and different DHCR24 heavy-expressing mice Dhcr24 hepKO in Example 5;

[0053] Fig.39 This is a schematic diagram of the process of treating DEN combined with CCL4 model liver cancer mice with irbesartan and olive oil control in Example 6;

[0054] Fig.40 This is a comparison chart of body weight, serum ALT and serum AST levels of liver cancer mice in the control group and the irbesartan treatment group in Example 6;

[0055] Fig.41 The WB images of liver tissues of liver cancer mice in the control group and the irbesartan treatment group in Example 6;

[0056] Fig.42 This is a comparison of liver cholesterol and liver 7-KC levels in the Veh control group and the Irbesartan treatment group of liver cancer mice in Example 6;

[0057] Fig.43 This is a comparison chart of tumor incidence, tumor number and tumor burden in the Veh control group and Irbesartan treatment group of liver cancer mice in Example 6;

[0058] Fig.44 The liver tissue photos and H&E staining microphotographs of the liver cancer mice in the control group and the irbesartan treatment group in Example 6;

[0059] Fig.45 is a WB image of three liver cancer cell lines after treatment with irbesartan or DMSO in Example 6;

[0060] Fig.46 From left to right are the cell index comparison diagrams of MHCC97H and HepG2 liver cancer cell lines after treatment with irbesartan or DMSO in Example 6;

[0061] Fig.47 This is a photo of cell clones formed by three liver cancer cell lines after treatment with irbesartan or DMSO in Example 6;

[0062] Fig.48 From left to right are comparison diagrams of cell clones of three liver cancer cell lines, MHCC97H, HepG2 and PLC5, after treatment with irbesartan or DMSO in Example 6;

[0063] Fig.49 These are WB images of two DHCR24 knockout cell models in Example 6;

[0064] Fig.50 From left to right are the cell index comparison diagrams of the two DHCR24 knockout cell models MHCC97H and SKHep1 in Example 6;

[0065] Fig.51 These are photos of cell clones formed by two DHCR24 knockout cell models in Example 6;

[0066] Fig.52 From left to right are comparison diagrams of cell clones of two DHCR24 knockout cell models MHCC97H and SKHep1 in Example 6. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme of the present invention is clearly and completely described in conjunction with the embodiments below, but it is not limited to this. Any modification or equivalent replacement of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention should be included in the protection scope of the present invention. The process equipment or devices not specifically specified in the following embodiments all adopt conventional equipment or devices in the field. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially, and the component raw materials used in each parallel experiment are of the same kind; if not specifically specified, the technical means used in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0068] The materials and methods used in the animal experiments of the present invention are as follows:

[0069] A. Human liver samples

[0070] Human normal liver and liver cancer tissue samples were collected from the Fourth Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all subjects, and the study protocol was approved by the Scientific and Clinical Research Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University.

[0071] B. Mouse Model

[0072] Mouse model of liver cancer

[0073] Wild-type (WT) mice were given a mouse carcinogen (Den) at 10-14 days of age after birth, with a single intraperitoneal injection at a concentration of 5 mg / kg. Carbon tetrachloride (CCL4) was injected intraperitoneally at 6 weeks of age, with a concentration of 0.2 ml / kg, twice a week, for 14 consecutive weeks. In the 7th week of CCL4 administration, the mice were randomly divided into two groups according to body weight and treated with olive oil or irbesartan (60 mg / kg / day). After 12 weeks of irbesartan treatment, the mice were killed to examine the therapeutic effect of irbesartan.

[0074] Hepatocyte-specific DHCR24 knockout mouse model

[0075] DHCR24 knockout mice were constructed by BIOCYTOGEN (Beijing, China) based on CRISPR / Cas9 technology.

[0076] To drive hepatocyte-specific knockout of DHCR24, DHCR24 KO mice were crossed with B6.CgTg(Alb-cre)21Mgn / JNju mice (Nanjing University, China). DHCR24 / Alb-Cre mice were verified by PCR genotyping.

[0077] For AAV modeling, 8-week-old control (Alb-Cre) and Dhcr24 hepKO male mice were infected by tail vein injection. For the expression of mouse Dhcr24 (NCBI reference sequence: NM_053272.2), 3 x 10 11 Each gene copy number was dissolved in PBS solution and AAV8-TBG-P2A-GFP (AAV-vector), AAV8-Dhcr24-WT, AAV8-Dhcr24-K254R, and AAV8-Dhcr24-K254Q were injected via tail vein as a single dose.

[0078] Assessment of Hepatocellular Carcinoma: Liver histology was assessed at sacrifice using H&E staining.

[0079] Inflammation assessment: Liver damage was determined using serum ALT and AST.

[0080] Nude mouse tumor model

[0081] A mouse model of xenograft human hepatoma cells was established using DHCR24 mutant stable cell lines (SKHep1 and MHCC97H). Human DHCR24 (NCBI reference sequence: NM_014762.4) mutants included NTC+EV, sg+EV, sg+WT, sg+K87R, sg+K87Q, sg+k254R, and sg+K254Q stable cell lines. Each cell (1.5×10 710 cells dissolved in 0.1 ml PBS) were injected subcutaneously into the left dorsal flank of 5-week-old male Balb / c nude mice. At the indicated time points, the mice were killed and examined.

[0082] All animal studies were performed according to the guidelines approved by the Animal Experiment Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University.

[0083] C. Preparation of acetylated antibodies (DHCR24-K87 and DHCR24-K254)

[0084] Polyclonal antibodies against acetylation of DHCR24 K87 or K254 (Q15392·DHC24_HUMAN) were generated according to the protocol of Pujian Biotechnology Co., Ltd. (Wuhan, China).

[0085] The simplified steps are as follows:

[0086] Two acetylated peptide-KLH antigens (EWKEQGSK(ace)TFMC for K87 and AK(ace)FTHESQRQEN+Cys for K254) were immunized in New Zealand white rabbits (4 conventional immunizations and 1-2 additional immunizations). Then, the indirect ELISA method was used to detect the antibody titer after the 3rd / 4th / 5th / 6th immunization using the screening peptides (EWKEQGSKTFMC for K87 and AKFTHESQRQEN+Cys for K254) as antigens. When the antibody titer exceeded 1:32000, the rabbit serum was collected and the antibody was purified using antigen-specific affinity.

[0087] D. Biological Function Analysis

[0088] Serum ALT and AST testing

[0089] A 5-μL blood sample was collected, and the ALT and AST levels were detected using an alanine aminotransferase activity detection kit (BC155, Solebao, China) and an aspartate aminotransferase activity detection kit (BC1565, Solebao, China), respectively.

[0090] E. Serum alpha-fetoprotein

[0091] Serum AFP was detected using an AFP ELISA kit according to the manufacturer's instructions (KE10092, Proteintech). 10 μL of mouse serum was diluted to 500 μL with sample diluent. The diluted samples, standards, and controls were then added to each microplate well for further analysis.

[0092] F. Histopathology and Immunohistochemistry

[0093] The samples were fixed in 4% paraformaldehyde for 24 hours at room temperature, then transferred to 70% ethanol for 12 hours, and then embedded in paraffin. Sections were cut using a microtome (Leica RM2235) and dewaxed by baking at 60°C for 3 minutes. The slides were then exposed to antigens. For immunohistochemical staining, endogenous peroxidase was inactivated with 3% hydrogen peroxide for 15 minutes at room temperature. To prevent nonspecific signals, the tissue was blocked with 5% BSA and 5% goat serum for 1 hour. The tissue was then incubated with the primary antibody (1:50 dilution) at 4°C for 12 hours. After washing with PBS-T, it was incubated with an anti-rabbit secondary antibody (1:1000 dilution) for 1 hour at room temperature.

[0094] G. Cell culture

[0095] HepG2, PLC5, and SKHep1 cells were purchased from ATCC (Manassas, VA). MHCC97H cells were provided by Professor Xiaoxing Li from Sun Yat-sen University, China.

[0096] The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 1% antibiotic-antimycotic (Anti-antimycotic; Gibco) and 10% fetal bovine serum (FBS Thermo Fisher Scientific). The cells were maintained at 37°C in a humidified incubator containing 5% CO2.

[0097] H.MTT proliferation assay

[0098] Cell proliferation activity was detected using the MTT method. Hepatocytes (SKHep1, HepG2, MHCC97H, and PLC5) were seeded in 96-well plates at 1500 cells / well and treated with Irbesartan or DMSO control. At the specified time point, 10 μL of MTT (5 mg / ml) solution was added to each well and cultured at 37°C for 4-6 hours. Then, all the culture medium was discarded and the cells were cultured with 100 μL DMSO in each well for 30 minutes. Finally, the cell growth curve OD490 was detected using a microplate reader.

[0099] I. Colony formation assay

[0100] Hepatocytes (SKHep1, HepG2, MHCC97H and PLC5) were placed in 6-well plates at 1500 cells / well and treated with Irbesartan or DMSO control. After 10-14 days of culture, cells were fixed with 70% ethanol and stained with 1% crystal violet solution for 15 minutes. The 6-well plates were washed with PBS and colony formation was counted.

[0101] J. Western blot analysis

[0102] Protein samples were first separated by SDS-PAGE. Then, the protein samples were transferred to nitrocellulose (NC) membranes (GE Healthcare). After blocking with 3% BSA buffer for 2 hours at room temperature, the membranes were incubated with primary antibody (1:1000 dilution in 1% BSA) overnight at 4°C, followed by incubation with secondary antibody (1:10000 dilution in 1% BSA) for 1 hour at room temperature. Finally, the membranes containing the proteins of interest were visualized using ECLPlus Western blotting detection reagent (GE Healthcare).

[0103] K. Statistical analysis

[0104] All statistical tests were performed using SPSS or GraphPad software. Data are expressed as mean ± SEM. Multiple group comparisons were analyzed by univariate ANOVA. Mann-Whitney U test or Student's t test was performed to compare variables between two groups. P value < 0.05 was considered statistically significant.

[0105] Example 1

[0106] This example verifies that the expression of 24-dehydrocholesterol reductase DHCR24 gene is significantly increased in liver cancer.

[0107] First, this example detected the expression of DHCR24 gene in two different liver cancer clinical cohorts (own cohort and Fudan cohort). Figure 1 As shown in the figure, A is the clinical cohort of liver cancer in the Fourth Affiliated Hospital of Harbin Medical University, n=10 pairs, and B is the clinical cohort of liver cancer in Fudan University, n=150 pairs; in these two clinical cohorts, compared with adjacent tissues, DHCR24 protein expression was significantly increased in liver cancer tissues. Figure 2 As shown, the samples were from the HMU (Harbin Medical University) liver cancer clinical cohort, and Western blot experiments confirmed that the expression level of DHCR24 gene protein was significantly increased in liver cancer tissues.

[0108] This example further analyzes the relationship between DHCR24 protein expression and the survival rate of liver cancer patients in clinic. Figure 3 These are the IHC color images of the gene chip at different DHCR24 protein expression levels; Figure 4 The relationship between DHCR24 protein expression level and clinical survival rate of liver cancer patients, DHCR24 protein high expression level group n = 59, DHCR24 protein low expression level group n = 29, combined with Figure 3 and Figure 4 Analysis showed that high DHCR24 protein expression levels were closely related to decreased survival rates of liver cancer patients in clinical practice.

[0109] Example 2

[0110] This example verifies that the acetylation modification level of the K254 site of the DHCR24 protein is increased in liver cancer, and the high acetylation level of the K254 site of the DHCR24 protein is positively correlated with the poor prognosis of the patient.

[0111] Since the mRNA expression of the DHCR24 gene remained unchanged in HCC (primary hepatocellular carcinoma) compared with non-tumor adjacent tissue control samples, while its protein expression was significantly upregulated in tumors, it was speculated that this difference was due to the presence of post-translational modification (PTM) of the DHCR24 gene in HCC.

[0112] In this example, acetylated proteomics sequencing was performed on HCC samples and non-tumor adjacent tissue control samples. The samples were from the HMU liver cancer clinical cohort, n = 10 pairs; the sequencing results are shown in Figure 5 As shown in Figure 3, DHCR24 protein has acetylation sites, and the acetylation level of K254 site in HCC is significantly higher than that in control tissues. Figure 6 As shown, the samples were from the HMU liver cancer clinical cohort, and Western blot experiments further confirmed that the acetylation level of the K254 site of the DHCR24 protein was significantly increased in liver cancer tissues.

[0113] This example further analyzes the relationship between the acetylation level of the K254 site of the DHCR24 protein and the survival rate of clinical liver cancer patients. Figure 7 This is the IHC color image of the gene chip with different acetylation levels of DHCR24 protein K254 site. Figure 8 The relationship between the acetylation level of DHCR24 protein K254 site and the survival rate of liver cancer patients in clinical practice was analyzed. The group with high acetylation level of DHCR24 protein K254 site was n=35, and the group with low acetylation level of DHCR24 protein K254 site was n=25. Figure 7 and Figure 8 Analysis showed that the acetylation level of K254 site of DHCR24 protein in HCC tissues was significantly increased, and a higher K254 acetylation level was associated with a poorer survival rate in clinical liver cancer patients.

[0114] Example 3

[0115] This example verifies that overexpression of the DHCR24 gene promotes the occurrence and development of liver cancer.

[0116] In order to study the function of the DHCR24 gene, this example established two cell models, SKHep1 and MHCC97H, that re-express DHCR24 based on knockdown of DHCR24. Fig. 9These are WB images of different SKHep1 and MHCC97H cell models, where sgControl represents cells that normally express DHCR24, sgDHCR24 represents cells in which DHCR24 expression is knocked down, and DHCR24 represents cells that express DHCR24 again after DHCR24 expression is knocked down.

[0117] from Fig.10 , Fig.11 and Fig.12 It can be seen from the comparison that DHCR24 overexpression promotes cell proliferation and clone formation. Therefore, this example confirms the cancer-promoting function of DHCR24 gene overexpression through the above cell test results.

[0118] In order to further evaluate the cancer-promoting function of DHCR24, this example constructed a hepatocyte DHCR24-specific knockout mouse (Dhcr24-hepKO). The mouse construction method and WB experiment are as follows: Fig.13 Wild-type Alb-Cre and Dhcr24hepKO mice were simultaneously given carcinogens (Den) and carbon tetrachloride (CCL4) to establish models, with intraperitoneal (IP) injections of DEN: 5 mg / kg body weight, single dose, and CCL4: 0.2 ml / kg body weight, twice a week, until the end. The modeling process is as follows Fig.14 shown.

[0119] like Fig.15 and Fig.16 As shown, DHCR24 knockout significantly reduced the incidence and number of HCC compared with the wild-type group. Fig.17 and Fig.18 The results showed that compared with wild-type control mice, inhibition of hepatocyte DHCR24 expression significantly reduced blood AFP, ALT and AST levels, but there was no significant difference in body weight between the two groups.

[0120] The above results once again confirmed that targeting DHCR24 is a feasible strategy for treating liver cancer and DHCR24 is a potential target for liver cancer treatment.

[0121] Example 4

[0122] This example verifies that acetylation at the K254 site of the DHCR24 protein stabilizes the protein level of DHCR24 and promotes its enzymatic activity and carcinogenicity.

[0123] In this example, stable cell lines overexpressing different DHCR24 mutant plasmids were generated to simulate acetylation in MHCC97H-sgDHCR24 and SKHep1-sgDHCR24 cells. KQ simulates acetylation at a specific site, and KR indicates that a specific site loses the ability to be modified by acetylation. The WB images of the stable cell lines overexpressing different DHCR24 mutant plasmids are shown in FIG. Fig.19 shown.

[0124] Chloroquine (CQ) is an inhibitor of the autophagy-lysosomal degradation pathway. In this example, 50 nM CQ was used to treat different DHCR24 mutant cells to observe the degradation level of DHCR24 protein. The results are as follows: Fig. 20 As shown, DHCR24 protein expression was increased in WT, K87Q, and K254R cells, but not in K254Q overexpressing cells. This suggests that acetylation of DHCR24 at K254 stabilizes protein levels in HCC.

[0125] Cycloheximide (CHX) can inhibit protein synthesis and is a common method for detecting the degradation level of specific proteins. In this example, 20 mg / ml CHX was used to treat different DHCR24 mutant cells to observe the degradation level of DHCR24 protein. The results are as follows: Figure 21-24 As shown, it was also confirmed that K254 acetylation significantly stabilized DHCR24 protein expression by inhibiting the degradation of DHCR24.

[0126] Since DHCR24 is a key enzyme in cholesterol biosynthesis, this example also detected the intracellular cholesterol level in the above stable cell lines. Fig.25 As shown, re-overexpression of DHCR24 greatly increased intracellular cholesterol levels. K254Q overexpression significantly increased intracellular cholesterol compared to K254R control, whereas there was no difference between K87Q and K87R stable expressing cells.

[0127] This example investigates the cell proliferation and clone formation of stable cell lines overexpressing different DHCR24 mutant plasmids. Figure 26-Figure 28 As shown, consistent with the alteration of cholesterol levels, K254R cells significantly inhibited cell proliferation and clonogenicity in HCC compared with DHCR24-WT and DHCR24-K254Q overexpression.

[0128] Overall, increased acetylation of DHCR24 at K254 stabilizes the protein and promotes its enzymatic activity and oncogenicity in HCC cell lines.

[0129] Example 5

[0130] This example verifies that increased acetylation of DHCR24 mediates the tumorigenic function of DHCR24 in vivo.

[0131] This example uses a nude mouse xenograft model to study the role of DHCR24 acetylation in the development of HCC. Stable cell lines with different DHCR24 variant expressions are transplanted by subcutaneous injection. For specific methods, see the nude mouse tumor formation rate model construction method. The observation period for tumor growth is 18 days. The results are shown in Figure 29-Figure 32 As shown, after transplantation of stable cell lines expressing different DHCR24 variants, knockdown of DHCR24 (sgDHCR24-Vector) significantly suppressed the weight and volume of subcutaneous SK-Hep1 xenograft-tumors and prolonged overall survival (tumor size 200 mm) compared with the control group (SgCtrl-Vector). 3 as the critical value).

[0132] Compared with the sgDHCR24-vector group (Vector), re-transfection of DHCR24-WT, K87Q, and K254Q plasmids into SKHep1-sgDHCR24 cells restored tumor weight and volume and worsened overall survival. However, overexpression of DHCR24-K87R and K254R plasmids resulted in loss of acetylation and did not significantly affect tumor volume, weight, or overall survival. These results preliminarily confirmed that increased acetylation levels are required for the tumorigenic activity of DHCR24 in HCC.

[0133] Next, this example used AAV technology to establish DHCR24 heavy-expressing mice Dhcr24 hepKO mice. For this method, 8-week-old Dhcr24 hepKO mice were injected with hepatocyte-specific adeno-associated virus 8 (AAV8) expressing different Dhcr24 variants, namely AAV8-TBG-P2A-GFP (AAV-vector), AAV8-Dhcr24-WT, AAV8-Dhcr24-K254R and AAV8-Dhcr24-K254Q through the tail vein. Control (Alb-Cre) and Dhcr24 hepKO mice were injected intraperitoneally (IP) with DEN: 5 mg / Kg body weight, single dose, CCL4: 0.2 ml / Kg body weight, twice a week until the end. The modeling process was as follows. Fig.33 shown.

[0134] like Figure 34-Figure 38 As shown, re-overexpression of hepatic DHCR24 in Dhcr24 hepKO mice restored carcinogen-induced HCC formation, as confirmed by restored tumor incidence and number, increased serum AFP, ALT, and AST levels, and increased serum and hepatic cholesterol and 7-KC (7-ketocholesterol) levels as well as macroscopic histology and H&E staining.

[0135] The injection of AAV-Dhcr24-K254Q to mimic the acetylation status of DHCR24 showed similar effects as Dhcr24-WT overexpression in restoring HCC formation. On the other hand, the loss of DHCR24 acetylation after AAV-Dhcr24-K254R injection significantly inhibited the development of HCC compared with Dhcr24 re-overexpression.

[0136] Taken together, these results suggest that elevated DHCR24 levels promote the occurrence and progression of HCC, depending on its increased acetylation at the K254 site.

[0137] Example 6

[0138] This example verifies that Irbesartan can inhibit liver cancer by inhibiting DHCR24 gene expression and acetylation.

[0139] This example first verifies the effectiveness of irbesartan in treating DEN combined with CCl4 model mice. The treatment process is shown in the schematic diagram. Fig.39 As shown, DEN: 5 mg / Kg body weight, single dose, CCL4: 0.2 ml / Kg body weight, twice a week were injected intraperitoneally (IP), until finally, irbesartan 60 mg / Kg body weight / once a day, the treatment period was 7 weeks.

[0140] like Fig.40 As shown in the results, irbesartan had no effect on the body weight of mice, while significantly reducing serum ALT and AST levels, indicating that it had no liver damage or toxicity to the liver. Fig.41 As shown in FIG. 1 , through Western blot experiments, this example found that irbesartan treatment significantly reduced the expression of DHCR24 protein in the liver and its acetylation level. Fig.42 As shown in Figure 2, irbesartan resulted in a decrease in hepatic cholesterol and 7-KC levels, indicating that the drug has an inhibitory effect on the enzymatic activity of DHCR24. Fig.43 As shown in Figure 2, irbesartan treatment also significantly reduced tumor incidence and tumor number. Fig.44 As shown in Figure 3, liver tissue images and HE results further confirmed this, showing that HCC tumor formation was significantly reduced after irbesartan treatment.

[0141] In summary, the above experimental results confirm that irbesartan, as a safe and effective DHCR24 inhibitor, has potential use in preventing and treating liver cancer.

[0142] After verifying that irbesartan can be used as a safe and effective DHCR24 inhibitor to prevent and treat liver cancer through the above animal experiments, this example then used irbesartan or DMSO to treat three liver cancer cell lines: HepG2, PLC5 and MHCC97H.

[0143] like Fig.45 Western blot results showed that irbesartan significantly inhibited the expression of DHCR24 protein level. At the same time, irbesartan effectively reduced the acetylation level of DHCR24 at K87 and K254 sites. Figure 46-Figure 47 As shown, irbesartan also significantly inhibited the cell proliferation and clone formation abilities of HepG2, PLC5 and MHCC97H cell lines.

[0144] Based on the above in vitro and in vivo experimental results, this example speculates whether irbesartan inhibits liver cancer in a DHCR24-dependent manner. Therefore, this example uses sgRNA to knock out endogenous DHCR24 and successfully constructs DHCR24 knockout cell models: MHCC97 and SKHep1.

[0145] like Fig.49 Western blot results showed that irbesartan significantly reduced the acetylation levels of DHCR24 at K87 and K254, but after knocking out endogenous DHCR24, irbesartan treatment did not significantly change the acetylation levels of DHCR24 at K87 and K254 compared with the sgDHCR24 control group. Figure 50-Figure 52 As shown, cell proliferation assay and clone formation assay also proved this point.

[0146] Based on the above results, this example believes that irbesartan exerts its effect of inhibiting liver cancer by relying on DHCR24. There is a mutual dependence between irbesartan and DHCR24 gene. Irbesartan can inhibit liver cancer by inhibiting DHCR24 gene expression and acetylation.

[0147] Example 7

[0148] This embodiment provides a liver cancer diagnosis kit, including a reagent for detecting the acetylation level of the K254 site of the DHCR24 protein. In this embodiment, the reagent for detecting the acetylation level of the K254 site of the DHCR24 protein is a specific antibody.

[0149] In this embodiment, the specific antibody is an acetylated polyclonal antibody against the K254 site of the DHCR24 protein. The polyclonal antibody is prepared by animal immunization and antibody separation and purification using the acetylated peptide-KLH as an antigen. The amino acid sequence of the acetylated peptide is AK(ace)FTHESQRQEN+Cys.

[0150] The preparation method of the acetylated polyclonal antibody of the K254 site of the DHCR24 protein in this example is as follows:

[0151] The acetylated peptide-KLH antigen was immunized in New Zealand white rabbits, with conventional immunization for 4 times and additional immunization for 2 times, and then the indirect ELISA method was used to detect the antibody titer after the 3rd, 4th, 5th and 6th immunizations with the screening peptide as the antigen; when the antibody titer exceeded 1:32000, the rabbit serum was collected and the antibody was purified using antigen-specific affinity; the amino acid sequence of the screening peptide was AKFTHESQRQEN+Cys.

Claims

1. 2 Application of the acetylation level of the K254 site of DHCR24 protein as a diagnostic marker in the preparation of a kit for diagnosis and prognosis assessment of liver cancer.

2. A kit for diagnosing liver cancer, characterized in that: Includes reagents for detecting the acetylation level of the K254 site of the DHCR24 protein.

3. A liver cancer diagnosis kit according to claim 2, characterized in that: The reagent for detecting the acetylation level of the K254 site of the DHCR24 protein is a specific antibody, a polypeptide or a small molecule preparation.

4. A liver cancer diagnosis kit according to claim 3, characterized in that: The specific antibody is an acetylated polyclonal antibody of the K254 site of the DHCR24 protein. The polyclonal antibody is prepared by using the acetylated peptide-KLH as an antigen through animal immunization and antibody separation and purification. The amino acid sequence of the acetylated peptide is AK(ace)FTHESQRQEN+Cys.

5. A liver cancer diagnosis kit according to claim 4, characterized in that: The preparation method of the acetylated polyclonal antibody of the DHCR24 protein K254 site is as follows: The acetylated peptide-KLH antigen was immunized in New Zealand white rabbits, with conventional immunization for 4 times and additional immunization for 2 times, and then the indirect ELISA method was used to detect the antibody titer after the 3rd, 4th, 5th and 6th immunizations with the screening peptide as the antigen; when the antibody titer exceeded 1:32000, the rabbit serum was collected and the antibody was purified using antigen-specific affinity; the amino acid sequence of the screening peptide was AKFTHESQRQEN+Cys.

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