Study On The Role Of DRAM1 In The Occurrence And Development Of NAFLD-related HCC

Objective:Non-alcoholic fatty liver disease(NAFLD)is the most common cause of chronic liver disease in Western countries,and the disease spectrum includes NAFL,NASH,cirrhosis,and hepatocellular carcinoma(HCC).Studies have shown that the annual incidence of HCC among NASH patients in the United States and Europe is between 0.7% and 2.6%,and the incidence of NAFLD-related HCC is expected to increase dramatically by 2030.However,there is still little research on this process.In this study,the expression level of DRAM1 in GEO data set and TCGA database was analyzed by bioinformatics technology.A transgenic mouse model of NAFLD-related HCC was constructed to explore the role of DRAM1 in the occurrence and development of NAFLD-related HCC.Methods:1.Bioinformatics analysis The gene expression matrix data of each group were downloaded by retrieving NAFLDrelated HCC GEO data set,and the gene expression matrix of TCGA patients with HCC,prognostic information and gene mutation data of HCC patients: DRAM1 expression analysis,immune infiltration analysis,survival analysis and gene mutation correlation analysis were performed respectively.To evaluate the role of DRAM1 as an indicator of the development and progression of NAFLD-related HCC.2.Construction of DRAM1 gene knockout or overexpression mouse models Liver-specific DRAM1 knockout mice were constructed using CRISPR-Cas9 technology combined with the Cre-Loxp system.Liver-specific DRAM1 overexpression transgenic mice were obtained by injecting adeno-associated viruses carrying hepatocellular specific promotors into the caudal vein of C57BL/6J wild mice aged 6-8 weeks.Construction of NAFLD-related HCC mouse model: The NAFLD-related HCC model was induced in transgenic mice fed a western diet(21.1% fat,41% sucrose,1.25% cholesterol and high glucose and fructose solution)and intrabitoneal injection of CCL4 once a week for 24 weeks.During model building,all mice were kept on a 12-hour light/dark cycle,freely fed water,and weighed twice a week.After building the model,each group of mice fasted for6 hours and weighed.After the mice were anesthetized,blood was taken from the eyeballs and then dissected.Observe and record the condition of each organ.Mouse livers were completely isolated,cleaned with normal saline,photographed and weighed.Liver tissue and plasma were stored separately in a-80℃ refrigerator.Mouse liver tissue was measured and part of liver tissue was taken for pathological section.Results:Through analysis in the TCGA database,DRAM1 expression in HCC patients was significantly increased(P < 0.0001),and ROC curves showed that DRAM1 was good at distinguishing between non-tumor patients and tumor patients(AUC = 0.784).The level of DRAM1 was positively correlated with the levels of CD8+ T cells,NK cells,B cells,monocytes and macrophages(P < 0.0001).By analyzing the relationship between the levels of macrophage markers and inflammatory cytokines and DRAM1 expression,the results were also positively correlated(P < 0.0001).According to the prognostic information of HCC patients,survival analysis was conducted using SPSS software,and the results showed that the survival time of DRAM1 low expression group was longer(P = 0.012).According to the mutation data of patients with liver cancer,R studio was used for gene mutation analysis.The results showed that DRAM1 expression was higher in the TP53 high expression group,and there was a positive correlation between the two expression levels(r= 0.529,P < 0.0001).DRAM1 expression was also significantly increased in NAFLDrelated HCC patients in the GSE164760 dataset(P < 0.01),and ROC curves showed that DRAM1 gene was also good at distinguishing between non-tumor patients and tumor patients(AUC = 0.652).Based on these results,we speculate that DRAM1 may be a potential biomarker for HCC or NAFLD-related HCC.By constructing NAFLD-related HCC gene knockout or overexpression mouse models,we found that the expression of the results of HE staining could detect significant HCC nodules after the modeling of NAFLD-related HCC.The results showed that the incidence of liver nodules in wild-type mice was significantly higher than that in DRAM1 knockout mice group,the maximum nodule diameter was larger than that in DRAM1 knockout mice group,and the incidence of HCC nodules was higher than that in DRAM1 knockout mice group.Masson staining and a-SMA immunohistochemistry showed that the degree of fibrosis in knockout mice was significantly lower than that in wild-type mice.The positive rate of Ki-67 in knockout mice was significantly lower than that in wild-type mice.In DRAM1 overexpressed mice,significant HCC nodules were observed in the liver of the overexpressed group,while no similar nodules were found in the control group.The number of liver cancer nodules and the incidence of HCC in the overexpression group were higher than those in the control group,and the degree of fibrosis and Ki-67 were also significantly higher than those in the control group.Conclusion:1.Bioinformatics analysis showed that DRAM1 expression was increased in tumor tissue samples from HCC and NAFLD-related HCC patients.2.Animal experiments showed that DRAM1 could promote the development of NAFLDrelated HCC.