| Background:Nonalcoholic fatty liver disease(NAFLD)is one of the most common causes of chronic liver disease worldwide.NAFLD is usually a progressive disease with variable course,ranging from simple steatosis(SS)to non-alcoholic steatohepatitis(NASH),which can eventually lead to cirrhosis and hepatocellular carcinoma(HCC).Currently,there is no reliable biomarker for the diagnosis of NAFLD and NASH and no specific therapies are approved.Dicer is best known for its role as a riboendonuclease,which belongs to RNase III family and specifically recognizes double stranded RNA.In the classic pathway of miRNA generation,after the primary miRNA(pri-miRNA)is cut into pre-miRNA by Drosha/DGCR8,Dicer processes it into an immature double stranded RNA molecule with a length of about 21-23 nucleotides,one of which is degraded to form a mature miRNA.MiRNA is an evolutionarily conserved small molecule involved in gene regulation.Down regulation of Dicer can cause miRNA imbalance.However,the roles of different miRNAs in the occurrence of NASH,NAFLD and HCC are still inconsistent.We hope to explore the molecular mechanisms of Dicer and its regulated miRNA in nonalcoholic fatty liver disease and hepatocellular carcinoma,so as to provide new ideas for its early diagnosis and clinical treatment.Objective:(1)To identify the pathological phenotypes of NASH and its related HCC after Dicer deletion in hepatocytes;(2)To study the effect of Dicer deficiency on miRNA and mRNA,and the possible molecular mechanism in NASH and its related HCC;(3)To investigate the effect of Dicer deletion on miR-192-3p/IGF2 and how to promote M1 polarization of macrophages.Methods:(1)Combined with TCGA database,GEO database and liver cancer tissue chip,different expression of Dicer in HCC and adjacent tissues was analyzed.(2)Based on the Cre-LoxP recombinase system,hepatocytes specific Dicer knockout mice were constructed.Dicer LKO mice and control mice were induced by intraperitoneal injection of DEN to construct HCC models.(3)Liquid chromatograph-mass spectrometry showed the difference of total lipid level in liver tissue between Dicer LKO mice and control mice.(4)Immunohistochemistry showed the related levels of increased lipogenesis and inflammatory cytokine in Dicer LKO mice and control mice,and levels of 4-HNE and MDA.(5)The levels of macrophage activation related cytokines by real-time PCR in liver tissues of Dicer LKO mice and control mice under basal status and after DEN treatments.Combined with the transcriptome data of HCC in TCGA database,the ratios of different macrophage types in Dicer high expression group and Dicer low expression group were compared.(6)RNA-seq and miRNA-seq showed the different expression of mRNA and miRNA in Dicer LKO mice and controls.Significantly different expression of was shown by real-time PCR.(7)Cell biology experiments were conducted to explore the effects of miRNA mimic on the mRNA and protein expression of related genes.(8)Cell biology experiments were conducted to explore the effects of overexpression miR-192-3p on macrophage M1 polarization.Results:Analysis of TCGA database showed that the expression of Dicer was significantly lower in cancer tissues of various adenoepithelial origins than in adjacent tissues.Immunohistorchemistry experiments showed that the expression of Dicer in tumor was significantly lower in adjacent tissues of liver cancer tissue microarray.Through the construction of Dicer LKO mice,we found that 9-month-old Dicer LKO mice had obvious lipid droplets accumulation in the cytoplasm of hepatocytes,inflammatory cell infiltration,mild hyperplasia of fibrous connective tissue,and even spontaneous hepatocellular carcinoma.The levels of serum ALT and ALP in Dicer LKO mice were significantly higher than control mice.After DEN treatments,Dicer LKO mice were more prone to induced HCC,and showed higher phospholipid levels by LC/MS.Lipid synthesis related factors ACLY,SREBP1,SREBP2 and lipid oxidation markers MDA and 4-HNE in the liver were significantly increased in Dicer LKO mice.No matter whether DEN treatment or not,macrophage M1 polarization was observed in liver tissue of Dicer LKO mice.RNA-seq showed that the expression of Igf2 in Dicer LKO mice was significantly higher than control mice.MiRNA-seq showed that a series of miRNAs such as miR-192-3p were significantly decreased in Dicer LKO mice.The target gene prediction of miR-192-3p showed that it has a negative regulatory effect of IGF2.When miR-192-3p mimic were transfected into Huh7 cells and HepG2 cells,the expression of IGF2 changed significantly at the mRNA and protein levels changed significantly.Condition culture of M0 type THP-1 cells found that overexpression of miR-192-3p can induce the macrophage M1 polarized.Conclusion:Dicer promotes the development of nonalcoholic fatty liver disease and hepatocellular carcinoma by mediated miR-192-3p increased IGF2. |
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