The Functions And Mechanisms Of NLRC3 In Non-alcoholic Steatohepatitis And Hepatocellular Carcinoma

Objective:To explore the functions and mechanisms of natural immune protein,NLRC3,in nonalcoholic fatty liver disease（NAFLD）,including nonalcoholic fatty liver（NAFL）,nonalcoholic steatohepatitis（NASH）and hepatocellular carcinoma（HCC）.Methods:Mice were used to construct a NAFL-NASH mouse model by feeding Western diet combined with intraperitoneal injection of CCl₄.And we collected human liver tissues from NASH,HCC and adjacent normal tissues.Western Blot,quantitative PCR（q PCR）and immunohistochemistry were used to detect expression level of NLRC3 in liver tissues of mice fatty liver and NASH model,and human NASH and HCC.Hu H-7 cells were transfected with si-RNA to construct NLRC3 low-expression cell lines,were stimulated by palmitic acid to construct NAFLD cell model,and were stimulated by IL-6 to simulate inflammation in NASH and HCC.Oil red O staining and intracellular triglyceride assay kit were used to estimate the function of NLRC3 in lipid deposition,and the expression of NLRC3 and gene related to lipid metabolism,inflammatory cytokiness and fibrotic factors was analyzed by q PCR.Ed U assay,cell scratch assay and Transwell invasion assay were used for assessing proliferation,migration and invasion,respectively.Flow cytometry and TUNEL assay were conducted to assess cell apoptosis.Western Blot was performed to detect signalling pathway related to NLRC3.Results:The expression of NLRC3 was reduced in human HCC tissues,compared with normal liver.There was found no difference of the expression of NLRC3 in mice of NAFL and NASH,and human NASH liver tissues,compared with control liver tissues.After NLRC3 knockdown in NAFLD cell model,there was no difference of lipid deposition,while the expression of key enzyme in lipid anabloism,ACC,was downregulated.And there was no differences of other key enzymes and transcription factors in lipid anabolism and catablism,and inflammatory cytokines.After NLRC3 knockdown,the expression of desmin was upregulated in the stimulation of IL-6.The results of Ed U,scratch assay and Transwell cell invasion assay showed that the proliferation,migration,and invasion were increased in Hu H-7 cells,after knocking down of NLRC3.And flow cytometry and TUNEL assay showed that Hu H-7 cell apoptosis was suppressed after NLRC3knockdown.As for the underlying mechanisms,knockdown of NLRC3 in Hu H-7 cells was associated with activation of IL-6/STAT3 pathway.Conclusion:The immune protein,NLRC3,may participate in the regulation of lipid metabolism,liver fibrosis and HCC progression,while not participate in the pathophysiology of lipid deposition,inflammation.In HCC,NLRC3 silencing in Hu H-7cells can promote the proliferation,migration and invasion of HCC cells,while inhibit the apoptosis.IL-6/STAT3 pathway activation may be the underlying mechanism for HCC when NLRC3 expression is silenced.Therefore,NLRC3 may play a protective role in HCC and might be a therapeutic target for the treatment of HCC.