The Role And Molecular Mechanism Of IRX3 In Non-alcoholic Fatty Liver Disease And Hepatocellular Carcinoma

Part I Irx3 Promotes Hepatic Lipid Accumulation and Liver Steatosis in MiceObjective:To investigate the role and molecullar mechanism of IRX3 in hepatic lipid accumulation.Methods:To upregulate or downregulate hepatic Irx3 expression, mice were respectively injected by cauda vein with Ad-Irx3, Ad-shIrx3 and Ad-gfp, and fed a chow diet or a high fat diet for 12 weeks. Hepatic histology, lipid ammulation and LD-associated proteins were detected in these mice. We downregulated the expression of Fsp27? or Perilipin 1 in AML12 transfected with Ad-Irx3, treated cells with 400?mol/l oil acid, and then detected intracellular lipid content. Furthermore, we use CHIP and Luciferase reporter assays to investigate the transcriptional regulation of Fsp27? or Perilipin 1 by Irx3.Results:Upregulation of Irx3 expression in liver promoted lipid accumulation and hepatic steatosis, increased the expressions of Fsp27? and Perilipin 1. On the contrary, downregulation of Irx3 resisted high fat diet induced liver steatosis and reduced the expressions of Fsp27? and Perilipin 1. However, the levels of other LD-associated proteins such as Cidea% Cideb?Atg1?Cgi-58 and Perilipin2-4 were no significant change. Compared to Ad-grp, Ad-Irx3 remarkably increased TG content in hepatocytes; however, knockdown of Fsp27? or Perilipin 1 in hepatocytes transfected with Ad-Irx3 lowered TG content. CHIP and luciferase reporter assays showed that Irx3 bound into the promoters of Fsp27? and Perilipin 1, and that Irx3 overexpression enhanced the promoter activities of Fsp27? and Perilipin 1.Conclusions:Irx3 promotes hepatic lipid accumulation and liver steatosis through transactivation of Fsp27? and Perilipin 1.Part ? The Functional Role and Regulatory Mechanism of IRX3 in Hepatocellular CarcinomaObjective:To investigate the functional role of IRX3 in Hepatocellular carcinoma (HCC) and explore the regulatory mechanism of IRX3 from an microRNA's perspective.Methods:QRT-PCR and Western blot were used to investigated the expression levels of IRX3 and miR-377 in two HCC cell lines (HepG2 and SMMC7721) and a normal liver cell line (LO2). Lentivirus was used to construct stable cell lines with overexpression or silence of IRX3 (HepG2-shIRX3 and SMMC7721-IRX3). CCK-8, colony formation, transwell and wound healing assays were performed to investigated the effects of IRX3 and miR-377 on HCC cell proliferation, migration and invasion. miR-377 mimics or Control mimics were transfected into HCC cells, then the mRNA and protein expressions of IRX3 were determined by qRT-PCR and Western blot. Luciferase reporter assay was performed to investigated whether miR-377 could directly bind to the 3'UTR of IRX3 mRNA.Results:We found that IRX3 was upregulated in HCC cell lines. Loss and gain of function studies revealed that IRX3 significantly promoted proliferation, migration and invasion of HCC cells in vitro. Western blot and luciferase reporter assay identified that IRX3 was a direct target of miR-377. In addition, miR-377 was downregulated in HCC cell lines, and overexpression of miR-377 inhibited HCC cells proliferation, migration and invasion. Moreover, miR-377 restoration significantly abrogated IRX3-induced proliferation, migration and invasion of HCC cells.Conclusions:IRX3 promotes HCC cell proliferation, migration, and invasion. IRX3 was a direct target of miR-377, and that downregulated miR-377 may contribute to the upregulation of IRX3 in HCC cells.