Application of DRAM1 as diagnosis and treatment target for resisting non-alcoholic fatty liver disease related hepatocellular carcinoma in preparation of kit, carrier and medicine

Abstract

The invention belongs to the technical field of biological medicines, and provides application of DRAM1 as a diagnosis and treatment target for resisting non-alcoholic fatty liver disease related hepatocellular carcinoma in preparation of a kit, a carrier and a medicine aiming at the problem of lack of effective treatment targets of NAFLD-related HCC. As a target spot for resisting the non-alcoholic fatty liver disease related hepatocellular carcinoma, the expression level of the DRAM1 in liver tissues of non-alcoholic fatty liver disease related hepatocellular carcinoma patients and mice is increased. According to the application disclosed by the invention, research is developed aiming at the effect of the DRAM1 in the generation and development of NAFLD-related HCC, the analysis of TCGA liver cancer queue data and a GSE164760 data set is taken as an entry point, the influence of the DRAM1 on the diagnosis and prognosis of the liver cancer is comprehensively analyzed, and verification is carried out through a mouse in-vivo experiment, so that a new thought and a new target spot are provided for clinical diagnosis and treatment of the liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of DRAM1 as a diagnostic and therapeutic target for non-alcoholic fatty liver disease-related hepatocellular carcinoma in the preparation of kits, carriers and drugs. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD), characterized by hepatocellular steatosis, may affect approximately 30% of the world's population. The spectrum of NAFLD includes simple fatty liver and non-alcoholic steatohepatitis (NASH), which may progress to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The incidence of NAFLD-related HCC (NAFLD-related HCC) is increasing annually worldwide. Diabetes, obesity, intestinal dysbiosis, and cirrhosis remain major risk factors for NAFLD-related HCC. High heterogeneity, a lack of biomarkers for early diagnosis and prognosis, high tumor resistance, a suppressive tumor immune microenvironment, and variable immunotherapy responses complicate the early diagnosis, treatment, and postoperative management of NAFLD-related HCC. To date, the main treatments for NAFLD-related HCC include surgical resection, liver transplantation, local ablation, chemotherapy, and immunotherapy. Studies have shown that immune checkpoint inhibitors are not suitable for the treatment of NAFLD-related HCC. Recently, several molecules have been reported to play important roles in the progression of NAFLD-related HCC. Laura et al. found that STARD1 deficiency in hepatocytes can alleviate NAFLD-related HCC by regulating the distribution of hepatic bile acids. Liu et al. found that squalene epoxidase can promote tumor growth in NAFLD-related HCC. However, the effectiveness of immunotherapy for NAFLD-related HCC remains low, and there is an urgent need to identify targets to improve the efficacy of immunotherapy for NAFLD-related HCC. Summary of the Invention

[0003] To address the lack of effective therapeutic targets for NAFLD-related HCC, the present invention provides the use of DRAM1 as a diagnostic and therapeutic target for non-alcoholic fatty liver disease-related hepatocellular carcinoma in the preparation of kits, vectors, and pharmaceuticals. This study investigates the role of DRAM1, a DNA damage-regulated autophagy regulator, in the development and progression of NAFLD-related HCC. Using data from the TCGA liver cancer cohort and the GSE164760 dataset as a starting point, the study comprehensively analyzes the impact of DRAM1 on liver cancer diagnosis and prognosis, validated through in vivo mouse experiments, and provides new insights and targets for clinical liver cancer diagnosis and treatment.

[0004] The present invention is achieved through the following technical solutions: On the one hand, the present invention provides the use of DRAM1 in the preparation of a kit for diagnosing, treating or prognosing non-alcoholic fatty liver disease-related hepatocellular carcinoma. DRAM1 is a target for treating non-alcoholic fatty liver disease-related hepatocellular carcinoma, and its expression level is increased in the liver tissues of patients with non-alcoholic fatty liver disease-related hepatocellular carcinoma and mice.

[0005] Furthermore, the kit comprises a reagent for detecting the expression level of DRAM1 gene or protein in a biological sample.

[0006] Furthermore, the reagent for detecting the expression level of DRAM1 gene in a biological sample includes specific primers and probes; furthermore, the expression level of DRAM1 is detected by real-time quantitative PCR, and the sequences of the specific primers are shown in SEQ ID NOs: 7 to 10.

[0007] Furthermore, the reagent for detecting the expression level of DRAM1 protein in a biological sample includes a specific antibody and a detection substrate.

[0008] Furthermore, the biological sample is blood, serum, exosomes or liver tissue biopsy sample.

[0009] In another aspect, the present invention provides a recombinant adeno-associated virus vector comprising a DRAM1 gene sequence.

[0010] Furthermore, the vector is inserted into the DRAM1 gene through the AgeI and XhoI restriction sites, and the target fragment is amplified using the primer pair SEQ ID NO: 1 and SEQ ID NO: 2.

[0011] In a third aspect, the present invention provides a drug for treating or inhibiting non-alcoholic fatty liver disease-related hepatocellular carcinoma, comprising a substance that knocks down DRAM1.

[0012] In a fourth aspect, the present invention provides a method for screening candidate drugs for non-alcoholic fatty liver disease-related hepatocellular carcinoma, comprising: testing candidate drugs in NAFLD-HCC cells or animal models with high DRAM1 expression; and selecting candidate drugs that can significantly reduce DRAM1 expression and inhibit tumor growth.

[0013] The present invention has the following beneficial effects: The present invention found that the expression of DRAM1 is significantly correlated with the expression of immune cells, and the expression level of DRAM1 is significantly increased in NAFLD-related HCC patients and mouse liver tissue. Mouse liver-specific knockout of Dram1 can alleviate liver fibrosis, damage and tumor conditions of NAFLD-related HCC. Mouse liver-specific overexpression of Dram1 promotes the progression of NAFLD-related HCC. The research results of the present invention indicate that DRAM1 may be a regulator of the pathogenesis of NAFLD-related HCC, and strategies aimed at inhibiting DRAM1 expression may have therapeutic potential for NAFLD-related HCC patients. The present invention provides a potential immunotherapy target for the treatment of NAFLD-related HCC, helps to improve the immunotherapy effect of NAFLD-related HCC, and helps to prepare NAFLD-related HCC drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 DRAM1 expression was analyzed for the TCGA database. (A) DRAM1 gene expression in healthy controls and HCC patients. (B) ROC curve and AUC values ​​for DRAM1 expression levels were used to distinguish HCC patients from healthy controls. P <0.05;** P <0.01; *** P <0.001; **** P< 0.0001.

[0015] Figure 2 Correlation analysis between DRAM1 expression and immune cell proportion for TCGA database. (AF) MCP-counter algorithm analysis of the correlation between DRAM1 expression level and immune cell proportion. (A) Correlation between DRAM1 expression level and T cell proportion. (B) Correlation between DRAM1 expression level and CD8 + (C) Correlation between DRAM1 expression level and monocyte proportion. (D) Correlation between DRAM1 expression level and NK cell proportion. (E) Correlation between DRAM1 expression level and B cell proportion. (F) Correlation between DRAM1 expression level and endothelial cell proportion. (G) Timer algorithm analysis of the correlation between DRAM1 expression level and immune cell proportion. P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001.

[0016] Figure 3 Correlation analysis of DRAM1 expression and macrophage marker expression levels for the TCGA database. (A) Correlation between DRAM1 expression and CD68. (B) Correlation between DRAM1 expression and CD163. (C) Correlation between DRAM1 expression and IL-1β. (D) Correlation between DRAM1 expression and IL-10.* P <0.05; ** P <0.01;*** P <0.001; **** P< 0.0001.

[0017] Figure 4 The relationship between HCC patient survival time and DRAM1 expression levels was analyzed for the TCGA database. (A) Survival analysis of HCC patients based on DRAM1 expression.* P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001.

[0018] Figure 5 Analysis of gene mutations in HCC patients using the TCGA database. Grouped by median DRAM1 expression levels: (A) Gene mutations in the low DRAM1 expression group. (B) Gene mutations in the high DRAM1 expression group. Grouped by median TP53 expression levels: (C) Comparison of DRAM1 gene expression levels between high and low TP53 expression groups. (D) Correlation between DRAM1 and TP53 gene expression levels. * P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001.

[0019] Figure 6 DRAM1 expression was analyzed for the GEO database. (A) DRAM1 gene expression in patients with NAFLD-related HCC and other groups combined. (B) ROC curve and AUC values ​​for DRAM1 expression levels distinguishing patients with NAFLD-related HCC and other groups combined. P <0.05; ** P <0.01;*** P <0.001; **** P< 0.0001.

[0020] Figure 7 This is the map of the gene cloning vector GV651: Figure 8 This is an agarose gel electrophoresis diagram. The DNA molecular weight standards from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, and 100 bp.

[0021] Figure 9 To verify the successful construction of liver-specific DRAM1 knockout mice and liver-specific overexpression mice at the mRNA level.

[0022] Figure 10 To verify the successful construction of liver-specific DRAM1 knockout mice and liver-specific overexpression mice at the mRNA level. (A) Differences in DRAM1 expression at the mRNA level in liver-specific DRAM1 knockout mice. (B) Differences in DRAM1 expression at the mRNA level in liver-specific DRAM1 overexpression mice. * P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001.

[0023] Figure 11 A liver-specific DRAM1 knockout mouse model for NAFLD-related HCC was constructed and relevant indicators were measured. (A) Difference in nodule number between wild-type mice and DRAM1 knockout mice. (B) Difference in maximum nodule diameter between wild-type mice and DRAM1 knockout mice. (C) Representative gross appearance and hematoxylin and eosin staining of the liver between the two groups (magnification 100×). (D) Difference in HCC incidence between wild-type mice and DRAM1 knockout mice. (E) Masson staining of wild-type mice and DRAM1 knockout mice (magnification 100×). (F) Immunohistochemistry of a-SMA in wild-type mice and DRAM1 knockout mice (magnification 100×). (G) Immunohistochemistry of Ki-67 in wild-type mice and DRAM1 knockout mice (magnification 100×). P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001. Figure 12 To construct a NAFLD-related HCC model in mice with liver-specific overexpression of DRAM1 and to detect related indicators.

[0024] (A) Difference in nodule number between control mice and mice with liver-specific overexpression of DRAM1. (B) Difference in maximum nodule diameter between control mice and mice with liver-specific overexpression of DRAM1. (C) Representative gross appearance and H&E staining of livers between the two groups (magnification 100×). (D) Difference in HCC incidence between control mice and mice with liver-specific overexpression of DRAM1. (E) Masson staining of control mice and mice with liver-specific overexpression of DRAM1 (magnification 100×). (F) Immunohistochemistry for a-SMA in control mice and mice with liver-specific overexpression of DRAM1 (magnification 100×). (G) Immunohistochemistry for Ki-67 in control mice and mice with liver-specific overexpression of DRAM1 (magnification 100×). * P <0.05; ** P <0.01; *** P <0.001; **** P< 0.0001. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Adeno-associated virus (AAV) vectors carrying a hepatocyte-specific promoter in the examples were purchased from Shanghai Gene Medical Technology Co., Ltd. Animal feed was purchased from Changzhou Shuyi Shuer Biotechnology Co., Ltd. All animal experimental procedures were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Ocean University of China. Kits for measuring hepatic triglycerides and total cholesterol were purchased from Nanjing Jiancheng Bioengineering Institute, China. Antibodies for α-smooth muscle actin (α-SMA) and Ki-67 for IHC were obtained from Sevier Biotechnology Co., Ltd. RNAiso and PrimeScript RT reagents were purchased from Takara, Japan. SYBR Green PCR reagents were purchased from Qiagen, Germany.

[0027] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels. Example

[0028] 1. Acquire and analyze the gene expression matrix data of the GSE164760 dataset in the GEO database and the liver cancer cohort data in the TCGA database. Specifically: 1.1 Retrieve GEO datasets related to NAFLD-related HCC. Search the website https: / / www.ncbi.nlm.nih.gov / geo / with the keyword “NAFLD-related HCC” to download the gene expression matrix for NAFLD-related HCC patients. Also, download the gene expression matrix for the liver cancer cohort, prognostic information for liver cancer patients, and gene mutation data from the TCGA website (https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga).

[0029] 1.2 DRAM1 expression in hepatocellular carcinoma was analyzed in GEO and TCGA datasets, and receiver operating characteristic (ROC) curve analysis was performed. ROC curves are used to determine the cutoff value, sensitivity, and specificity of the indicator. The area under the ROC curve (AUC) is used to assess the diagnostic accuracy of the indicator. The results showed that DRAM1 gene expression was significantly increased in patients with liver cancer, with a statistically significant difference ( P <0.0001) ( Figure 1 A). By analyzing the AUC value of the receiver operating characteristic curve of the DRAM1 gene in the liver cancer cohort, it was found that the expression level of the DRAM1 gene can well distinguish between tumor and non-tumor patients (AUC = 0.784) ( Figure 1 B).

[0030] 1.3 The relationship between DRAM1 expression and immune cell infiltration ratio was analyzed by MCP-counter algorithm to explore the effect of DRAM1 gene on immune infiltration in HCC patients. The results showed that the expression level of DRAM1 gene was significantly correlated with T cells (r = 0.5005) ( Figure 2 A), CD8+ T cells (r = 0.3804) ( Figure 2 B), Monocytes (r = 0.4749) ( Figure 2 C), NK cells (r = 0.4466) ( Figure 2 D), B cells (r = 0.4909) ( Figure 2 E) and endothelial cells (r = 0.2518) ( Figure 2 F) expression levels were positively correlated, and the results were statistically significant ( P< 0.0001).

[0031] The expression of DRAM1 and the proportion of immune cell infiltration were analyzed by the Timer algorithm ( Figure 2G), the results showed that the expression level of DRAM1 gene was positively correlated with the expression levels of B cells (r = 0.507), CD8+ T cells (r = 0.444), CD4+ T cells (r = 0.460), macrophages (r = 0.594), neutrophils (r = 0.483), and dendritic cells (r = 0.623), and the results were statistically significant ( P< 0.0001).

[0032] 1.4 The expression level of DRAM1 in HCC tumor tissue is positively correlated with the expression level of macrophage markers.

[0033] By analyzing the expression of macrophage surface markers and related inflammatory genes, the correlation between the expression level of DRAM1 and macrophages was further explored. The results showed that the expression level of DRAM1 was significantly correlated with CD68 (r = 0.4126) ( Figure 3 A), CD163 (r = 0.4018) ( Figure 3 B), IL-1β (r = 0.5770) ( Figure 3 C), IL-10 (r = 0.5020) ( Figure 3 D) expression levels were positively correlated, and the results were statistically significant ( P <0.0001).

[0034] 1.5 We downloaded the prognostic data of HCC patients from the TCGA database. We divided the HCC patients into quartiles based on the expression of DRAM1. We used SPSS software to perform Kaplan-Meier survival analysis to explore the effect of DRAM1 gene expression on the prognosis and survival of HCC patients. The results showed that patients in the low DRAM1 gene expression group had a longer survival time than those in the high DRAM1 gene expression group ( P = 0.012)( Figure 4 A).

[0035] 1.6 Correlation between DRAM1 expression and TP53 mutation and expression in HCC tumor tissues. Previous studies have shown that P53 is an upstream gene of DRAM1 and that in mild steatosis, P53 promotes DRAM1 expression and induces autophagy, thereby inducing cell apoptosis. Therefore, we downloaded the gene mutation data of liver cancer patients from TCGA and grouped them according to the median DRAM1 expression level to compare gene mutations between the two groups. The results showed that in the high DRAM1 expression group ( Figure 5 B), the mutation rate of TP53 gene was the highest (38%), while in the low DRAM1 expression group ( Figure 5A), TTN gene mutation rate was the highest (26%). TP53 expression was analyzed, and the groups were divided according to the median expression level. The expression of DRAM1 gene was compared between the two groups. It was found that the TP53 high expression group had a higher expression level of DRAM1, and the results were statistically significant ( P <0.0001) ( Figure 5 C). Correlation analysis of TP53 expression and DRAM1 expression revealed a significant correlation between the two (r = 0.529) ( Figure 5 D), and there was a statistical difference ( P <0.0001).

[0036] 1.7 DRAM1 is highly expressed in NAFLD-related HCC patients. By searching the NAFLD-related HCC dataset (GSE164760) in the GEO database and analyzing it, the results showed that the expression of DRAM1 gene was significantly increased in the NAFLD-related HCC Tumor group, with a significant statistical difference ( P <0.01) ( Figure 6 A). We also analyzed the AUC value of the receiver operating curve of the DRAM1 gene in NAFLD-related HCC patients ( Figure 6 B) It was found that the expression level of the DRAM1 gene could well distinguish NAFLD-related HCC Tumor and other NAFLD-related HCC groups (AUC = 0.652).

[0037] 2. Construction, packaging, purification, and titer determination of recombinant adeno-associated viral vectors. The AAV construction process is as follows: Cloning the exogenous gene into a suitable viral vector: The exogenous gene is cloned into a vector containing the ITR / MCS. The inverted terminal repeat (ITR) sequences in these vectors provide all the cis-acting elements necessary for AAV replication and packaging. The recombinant expression plasmid is co-transfected into AAV-293 cells along with pHelper (carrying the adenoviral-derived genes) and pAAV-RC (carrying the AAV replication and capsid genes) (providing the trans-acting factors required for AAV replication and packaging). Two days after transfection, recombinant AAV is assembled in the packaging cells. AAV particles are harvested from the infected AAV-293 cells. AAV particles are generally concentrated in the packaging cells, so harvesting the cells and then lysing the cells to release the AAV particles into the supernatant allows for the recovery of the majority of the AAV particles. The viral supernatant, which contains numerous cellular proteins and debris, was concentrated and purified. Two cycles of CsCl density gradient centrifugation and one cycle of ultrafiltration removed most of the cellular proteins and residual CsCl ions. Finally, the titer of the resulting virus was determined using quantitative PCR.

[0038] 2.1 Vector Preparation Vector name: GV651 Element sequence: pAAV-ApoE / hAATp→MCS→EGFP-3Flag→SV40 PolyA→ITRs Cloning sites: AgeI and XhoI.

[0039] Vector length: 5.0kb.

[0040] 2.2 Obtaining target gene fragments (DRAM1) Primer design: Amplification primers are shown in Table 1.

[0041] Table 1. DRAM1 primers

[0042] PCR product size: 761 bp. Electrophoresis verification showed a band around 750 bp, such as Figure 8 shown.

[0043] 2.3 Vector digestion and ligation Double-digest GV651 with AgeI and XhoI to linearize the vector and expose sticky ends. Verify complete digestion by electrophoresis. Digest the PCR product (761 bp) with the same enzymes, purify it, and ligate it into the vector.

[0044] 2.4 Recombinant plasmid construction Use T4 DNA ligase to ligate the digested GV651 and DRAM1 fragments. Theoretical recombinant plasmid size: 5.0 kb + 761 bp = 5.761 kb. Transform the ligated product into competent cells (e.g., DH5α) and screen for positive clones by coating plates containing antibiotics (e.g., Amp).

[0045] 2.5 Identification of positive transformants

[0046] Table 2. PCR identification primers

[0047] The size of the PCR product of the positive transformant was 826 bp.

[0048] Analysis of electrophoresis results: Successful cloning, with bands located between 750 bp and 1 kb, as shown in Figure 9 shown.

[0049] Electropherogram Description: 1#: negative control (ddH2O); 2#: negative control (empty self-ligation control group); 3#: positive control (GAPDH); 4#: DNA molecular weight standard bands from top to bottom are 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, 100 bp; 5-12#: Transformants No. 1-8.

[0050] 3. AAV vectors carrying a hepatocyte-specific promoter were used to specifically knockout or overexpress DRAM1 in mouse hepatocytes. Dram1-flox mice were constructed using CRISPR-Cas9 combined with Cre-Loxp genetic engineering techniques, and mated to obtain homozygous and wild-type mice expressing the DRAM1 gene. Tables 3 and 4 detail the sgRNA sequences and primers used to construct and characterize Dram1-flox C57BL / 6J mice. Dram1-flox mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. and injected via the tail vein with an AAV vector carrying the Cre enzyme (pAAV-ApoE / hAATp-Cre) or a negative control vector (pAAV-ApoE / hAATp-null). Six-week-old male C57BL / 6J mice were injected via the tail vein with an AAV vector carrying the Dram1 gene (pAAV-ApoE / hAATp-Dram1) or a negative control vector (pAAV-ApoE / hAATp-null) to generate liver-specific overexpression of the DRAM1 gene. Real-time quantitative PCR was used to verify DRAM1 mRNA expression. The PCR amplification procedure is shown in Table 5.

[0051] Table 3. Construction of Dram1-flox mouse sgRNA

[0052] Table 4. Primers for genotyping Dram1-flox mice

[0053] Table 5. PCR amplification procedure for genotyping DRAM1 transgenic mice

[0054] The mRNA expression level of DRAM1 gene in mouse liver tissue was detected.

[0055] 3.1 Place 10 mg of mouse liver in a 1.5 mL EP tube. Add 1 mL of Trizol to lyse the liver tissue and grind with a sterile grinding rod for 2-3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Remove the supernatant and transfer 0.5 mL of the supernatant to new, sterile, RNase-free 1.5 mL EP tubes. Store one tube at -80°C, and use the other for subsequent experiments. Add chloroform to the tube at a volume ratio of 1:5 chloroform:supernatant. Mix by inversion for 15 seconds until the tube turns pink. Let stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Remove the supernatant and add isopropanol at a volume ratio of 1:1. Mix by inversion. Let stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. Remove the supernatant and add isopropanol at a volume ratio of 1:1. Mix by inversion. Let stand at room temperature for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C. Remove the supernatant and wash the tube with 1 mL of 75% ethanol (750 μL of anhydrous ethanol + 250 μL of DEPC water). Vortex the tube to wash, then centrifuge at 12,000 rpm for 5 minutes at 4°C. After removing the supernatant, dry the EP tube in a desiccator for 15 minutes (optionally on ice). Then, add 30 μL of DEPC water to dissolve the precipitate. Add 4 μL of RNA to 996 μL of single-distilled water, mix thoroughly by pipetting, and measure RNA concentration using a spectrophotometer.

[0056] 3.2 Reverse transcription was performed using the PrimeScript TMRT kit to generate cDNA. Real-time fluorescence quantitative PCR was then performed using the SYBR Green PCR kit with -actin as an internal control to detect DRAM1 gene expression. The primers used for RT-PCR are shown in Table 6.

[0057] Table 6. Primer sequences used in RT-PCR

[0058] The results are as follows Figure 10 As shown, liver-specific DRAM1 gene knockout mice and liver-specific overexpression mice were successfully constructed.

[0059] 4. Construction of mouse model and study of DRAM1 gene function.

[0060] 4.1 Eight-week-old C57BL / 6J male mice (containing the DRAM1 transgenic model) were randomly divided into two groups according to their genotype: a wild-type group (5 mice) and a DRAM1 knockout group (3 mice), and a wild-type group (10 mice) and a DRAM1 overexpression group (6 mice). They were fed a Western diet (WD) containing 21.1% fat, 41% sucrose, and 1.25% cholesterol and a high-glucose solution (Solar Bio) containing 23.1 g / L d-fructose and 18.9 g / L d-glucose for 24 weeks. At the same time, CCL4 (0.25 μg g -1 week -1 ) for 24 weeks to induce a NAFLD-related HCC model. A control diet (CD) was used. All mice were housed under conditions of temperature (22°C) and humidity (40%-60%), with a 12-hour light-dark cycle, free access to food and water, and body weight was measured twice weekly.

[0061] 4.2 After model establishment, mice in each group were deprived of food and water starting at 7:00 AM and weighed 6 hours later. After anesthesia, blood was drawn from the mice's eyeballs, and the mice were then dissected. The condition of each organ was observed and recorded. The liver was completely isolated, washed with saline, photographed, and weighed. Liver tissue and plasma were stored in separate tubes at -80°C for subsequent experiments.

[0062] 4.3. Liver pathology and biochemical index detection The diameter of the largest cross-section of the tumor was measured, and the mouse livers were sliced ​​at 1 mm intervals. The number of tumors at each layer was counted, and the number and size of liver cancer nodules, as well as the incidence of HCC, were statistically analyzed in both groups. Liver damage, steatosis, fibrosis, and tumor progression were verified at the histological and biochemical levels.

[0063] Lipid metabolism indicators: kits were used to measure liver triglycerides (TG) and total cholesterol (TC); automatic biochemical analyzer was used to detect plasma liver injury markers TG, TC, ALT, and AST.

[0064] Tissue staining: Oil Red O staining: Frozen sections demonstrate lipid droplet deposition in hepatocytes (steatosis). Masson staining / Sirius Red staining: Paraffin sections demonstrate collagen fibers (assessing liver fibrosis). H&E staining: Routine pathological observation (inflammation, necrosis, and tumor architecture). Immunohistochemistry (IHC): Detects α-SMA (a marker of fibrosis) and Ki-67 (a marker of proliferation).

[0065] like Figure 11Figure C shows liver-specific DRAM1 knockout mice. Based on the mouse liver and pathological findings, Figure C shows nodules in the liver of wild-type mice. HE results showed clear cell-like changes, indicating HCC-like nodules. Similar nodules were not found in homozygous mice. HE results of liver tissue from the same location showed significant fatty degeneration, but no HCC lesions were observed. Statistical analysis was also performed on the number, size, and HCC incidence of liver cancer nodules in the two groups ( Figure 11 AB, D), the number of liver cancer nodules in homozygous mice ( P <0.05) and the maximum nodule diameter ( P <0.05) were lower than those in wild-type mice, and the incidence of HCC was also significantly lower than that in wild-type mice ( P <0.05). Masson staining and a-SMA immunohistochemistry ( Figure 11 EF), found that the degree of fibrosis in homozygous mice was significantly lower than that in wild-type mice. Figure 11 G) The Ki-67 positive rate in homozygous mice was significantly lower than that in wild-type mice.

[0066] like Figure 12 As shown in c, in mice with liver-specific overexpression of DRAM1, HCC-like nodules were found in the liver tissues of both groups based on the mouse liver and pathological results. Figure 12 The number of liver cancer nodules in DRAM1-overexpressing mice was found in AB, D ( P <0.05) were higher than those in the control group, and the incidence of HCC was also significantly higher than that in the control group ( P <0.05). Masson staining and a-SMA immunohistochemistry ( Figure 12 EF), found that the degree of fibrosis in DRAM1-overexpressing mice was significantly higher than that in control mice. Similarly, Ki-67 ( Figure 12 G) The Ki-67 positive rate in DRAM1-overexpressing mice was significantly higher than that in the control mice.

[0067] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Use of DRAM1 in the preparation of a kit for diagnosing, treating or prognosing non-alcoholic fatty liver disease-related hepatocellular carcinoma, characterized in that: DRAM1 is a target for anti-nonalcoholic fatty liver disease-related hepatocellular carcinoma, and its expression level is increased in the liver tissues of patients with nonalcoholic fatty liver disease-related hepatocellular carcinoma and mice.

2. The use according to claim 1, characterized in that The kit comprises a reagent for detecting the expression level of DRAM1 gene or protein in a biological sample.

3. The kit according to claim 2, wherein The reagent for detecting DRAM1 gene in biological samples includes specific primers and probes; the reagent for detecting DRAM1 gene in biological samples includes specific primers and probes; 1. Protein expression level reagents include specific antibodies and detection substrates.

4. The kit according to claim 3, wherein The expression level of DRAM1 was detected by real-time quantitative PCR, and the specific primer sequences were shown in SEQ ID NOs: 7-10.

5. The kit according to claim 1, wherein The biological sample is blood, serum, exosomes or liver tissue biopsy sample.

6. A recombinant adeno-associated virus vector, characterized in that: Contains the DRAM1 gene sequence.

7. The recombinant adeno-associated virus vector according to claim 5, characterized in that The vector is inserted into the DRAM1 gene via the AgeI and XhoI restriction sites, and the target fragment is amplified using the primer pair SEQ ID NO: 1 and SEQ ID NO:

2.

8. A drug for treating or inhibiting hepatocellular carcinoma associated with non-alcoholic fatty liver disease, characterized in that: Contains a substance that knocks down the DRAM1 gene.

9. A method for screening candidate drugs for treating non-alcoholic fatty liver disease-related hepatocellular carcinoma, characterized in that: include: Testing drugs in NAFLD-HCC cells or animal models with high DRAM1 expression; Select drugs that can significantly reduce DRAM1 expression and inhibit tumor growth.

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