Inhibitory Effects And Molecular Mechanisms Of NDRG2 Targeting ACC1 To Inhibit The De Novo Synthesis Of Fatty Acids In Hepatocellular Carcinoma

Hepatocellular carcinoma is one of the common malignant tumours worldwide and ranks fifth in incidence and second in mortality in China,with a high proportion of primary hepatocellular carcinoma.NDRG2 is a downstream regulator of N-Myc gene 2,which can inhibit tumour development and progression in many ways.Objective:To observe the regulation mode and regulatory characteristics of genen N-Myc downstream regulatory gene 2（NDRG2）on lipid metabolism in human hepatocellular carcinoma cells,and mainly to study the regulatory role of NDRG2 on ACC1,a key rate-limiting enzyme of de novo lipogenesis,and elaborate the role of NDRG2 in regulating of ACC1 and de novo lipogenesis in hepatocellular carcinoma.Methods:To collect NDRG2 expression pattern in various hepatocellular carcinoma cells based on public bioinformatics website and screen cell lines.NDRG2 overexpression cells were obtained by infecting human hepatocellular carcinoma cell line Hep G2 with lentivirus carrying NDRG2 c DNA or control lentivirus,respectively,and the m RNA content of NDRG2 gene in the two groups of cells was measured by real-time quantitative PCR;The protein level of NDRG2 in cells was measured by Western-blot.NDRG2^-/-and NDRG2^+/+mouse embryonic fibroblasts（MEF）were extracted and the NDRG2 protein and m RNA levels were measured.The NDRG2 expreesion in the liver tissues of mice of both genotypes were detected by Western-blot and RT-PCR experiments.Metabolomics assays were performed to verify the regulatory effect of NDRG2 on lipid metabolism in NDRG2 knockout mice and NDRG2 overexpressing hepatocellular carcinoma cells.The content of phospholipids in NDRG2 overexpressing hepatocellular carcinoma cells were examined using enzyme-linked immunoassay kits,and the contents of triglyceride in NDRG2 overexpressing hepatocellular carcinoma cells were detected by oil red staining.Indirect immunofluorescence assay was used to detect the localization of NDRG2 and ACC1 proteins in hepatocytes.Immunohistochemical assay was used to detect analyze the expression of NDRG2 and ACC1 in tissue microarrays containing 57 hepatocellular carcinoma and adjacent normal hepatic tissues.The relationship between NDRG2 and ACC1 expression abundance and prognosis in hepatocellular carcinoma patients was analyzed by bioinformatics analysis through THPA（The Human Protein Atlas）databases.Results:The results of the bioinformatics data showed that NDRG2 was high expressed in Hep G2 cells and could be used to analyze the biological functions of NDRG2 in hepatocellular carcinoma cell.Metabolomic studies revealed that NDRG2 deficiency in normal mouse liver tissues resulted in hyperlipid synthesis,while enhanced NDRG2 expression significantly regulated the content of lipids in hepatocellular carcinoma cells,with significant changes in phospholipids:lecithin PC,phosphatidylglycerol PG,phosphatidylethanolamine PE.Enzyme-linked immunoassay revealed that enhanced expression of NDRG2 significantly suppressed the content of various phospholipid metabolites in the hepatocellular carcinoma cells Hep G2.Our previous TAP-MS data indicated that ACC1,the key enzyme of de novo lipogenesis,possibly interacted with NDRG2.Our current research results indicated that:NDRG2 overexpression inhibited ACC1 protein level in hepatocellular carcinoma cells analyzed by Western-blot;The results of indirect immunofluorescence experiments demonstrated that both NDRG2 and ACC1 were co-localized in the cytoplasm,maybe there exists an interaction between NDRG2 and ACC1.Immunohistochemical results showed there exists a negative correlation between the expression of NDRG2 and ACC1 in hepatocellular carcinoma tissues.The patients with high NDRG2 expression survived longer,and those with low ACC1 expression survived longer,suggesting that NDRG2 and ACC1 exhibit opposite effects on the prognosis of hepatocellular carcinoma patients.Conclusion:Database analysis revealed that NDRG2 expression levels were significantly decreased in hepatocellular carcinoma tissues,while patients with high NDRG2 expression had longer overall survival and patients with hepatocellular carcinoma with high ACC1expression had significantly poorer survival expectations than those with low expression,suggesting that NDRG2 and ACC1 may be significant independent prognostic indicators for hepatocellular carcinoma.Experimental studies demonstrated that:1.NDRG2 may have a regulatory role on lipid metabolism in tumor cells;2.The co-localization results of NDRG2 and ACC1 indicated that the two may interact in the cytoplasm,and NDRG2 overexpression could inhibit ACC1 protein content;3.NDRG2 could regulate phospholipid and triglyceride and phospholipid content in hepatocellular carcinoma cells through ACC1,and inhibit the occurrence and development.