The Role And Mechanisms Of Micro RNAs In Non-alcoholic Fibrosing Steatohepatitis

Nonalcoholic fatty liver disease(NAFLD) is a hepatic manifestation of the metabolic syndrome without excessive alcohol intake and other definite etiological factors, and is characterized by hepatocyte steatosis. NAFLD has became one of the most common chronic liver diseases. Nonalcoholic steatohepatitis(NASH) is the key stage of NAFLD and is characterized by non-alcoholic steatohepatitis and/or fibrosing steatohepatitis. Without appropriate intervention, NASH can progress to cirrhosis and even hepatocellular carcinoma. However, the pathogenesis of non-alcoholic fibrosising steatohepatitis remains poorly understood and definitive diagonostic methods and therapies for NASH patients are lacking.Micro RNAs(mi RNAs) are a class of short non-coding RNAs, about19-22 nucleotides in length, that can bind to the 3’-untranlated regions(3’UTR) in target m RNA molecules, causing translation repression or the cleavage of the target m RNAs. Many studies demonstrated that mi RNAs play essential roles in a variety of cellular processes such as metabolism, immune function, cell proliferation, and apoptosis. Dysregulation of mi RNAs may contribute to the development of NAFLD. However, the roles and mechnisms of mi RNAs in the pathogenesis of non-alcoholic fibrosing steatohepatitis are not fully understood.Heme oxygenase-1(HO-1), the rate-limiting enzyme in heme catabolism,has been reported to have potential antioxidant properties. HO-1 plays a pivotal role in non-alcoholic fibrosing steatohepatitis. However, the protective mechanism of HO-1 on non-alcoholic fibrosing steatohepatitis remains unclear. Whether HO-1 regulates the expression of mi RNAs in the development of non-alcoholic fibrosing steatohepatitis is still unknown.In this study, we established a non-alcoholic fibrosing steatohepatitismodel by feeding mice with methionine and choline deficient(MCD) diet.Using microarray assay, bioinformatics, dual-luciferase reporter gene assay,primary cell isolation, and si RNA transfection, we explored the aberrant expression of mi RNAs profile, and the role and mechanism of mi RNA in non-alcoholic fibrosing steatohepatitis. We further investigated the effect of HO-1 on mi RNA and its molecular mechanism on the progression of non-alcoholic fibrosing steatohepatitis in order to provide a basis for NAFLD prevention and the development of mi RNA targeting drugs.Part 1 Screening and validation of hepatic micro RNA profile in non-alcoholic fibrosing steatohepatitis in miceObjective: To establish a mouse model of non-alcoholic fibrosing steatohepatitis, screen and validate the expression profiles of dysregulated mi RNAs in the liver.Methods: Animal model of non-alcoholic fibrosing steatohepatitis was established by feeding mice with methionine-choline deficient(MCD) diet for8 weeks. Mice fed with choline-methionine supplemented diet were used as controls. Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were analyzed using enzymic method by automatic biochemistry analyzer. Hematoxylin and eosin(H&E) and Masson staining were performed to evaluate hepatic inflammation and fibrosis, respectively.The stage of steatosis and fibrosis was assessed according to the guidelines for diagnosis and treatment of non-alcoholic fatty liver diseases. Hepatic fibrogenic genes were measured by Western blot analyse. Total RNAs from mice liver tissue were isolated using Trizol reagent, and the quality of extracted RNA was evaluated. The mi RNA profiles were determined using micro RNA Microarray-20.0 Mouse(LC Sciences). quantitative real-time PCR(q RT-PCR) was performed to validate the result of microarray.Results:1 Serum transaminases changes: A significant increase in both ALT and AST was observed in the MCD diet fed mice, as compared to the control mice.2 Liver histology: H&E and Masson staining of liver sections from MCD fed mice showed disordered lobule structure, severe macrosteatosis, spot or focal necrosis, inflammatory infiltration, and portal and perisinusoidal fibrosis at week 8. However, liver sections of the control mice showed normal histopathological characteristics.3 Hepatic expression of fibrogenic genes: Western blot showed that the hepatic levels of transforming growth factor β1(TGF-β1), α-smooth muscle actin(α-SMA), Smad4, Collagen Type 4(Col-4), and matrixmetalloproteinase-2/9(MMP-2/9) were significantly increased while the expression of tissue inhibitor of metalloproteinase 1(TIMP 1) and Smad7 were significantly decreased in MCD fed mice, all relative to controls.4 The recults of micro RNA array: The microarray showed that 37 mi RNAs in the liver tissues of MCD fed mice or control diet fed mice were deregulated, of which 19 mi RNAs were up-regulated in MCD group(including mi R-15b-5p, mi R-150-5p, and mi R-106a-5p) and 18 mi RNAs were down-regulated(including mi R-146a-5p, mi R-203-3p, and mi R-130a-3p).5 Validation of dysregulated mi RNAs: q RT-PCR was performed to validate the expression of mi R-15b-5p, mi R-146a-5p and mi R-203-3p. It was found that the expression of mi R-15b-5p was up-regulated while the expression of mi R-146a-5p and mi R-203-3p was significantly down-regulated in the livers of MCD fed mice as compared to the control mice(P < 0.05).Conclusions:1 Non-alcoholic fibrosing steatohepatitis was successfully induced in mice fed with MCD diet for 8 weeks.2 Significant changes in the hepatic expression of mi RNAs were observed in mice with non-alcoholic fibrosing steatohepatitis, suggesting a pathogenic role of mi RNAs in the development of non-alcoholic fibrosing steatohepatitis.3 There was a significant down-regulation of hepatic mi R-146a-5p in mice with non-alcoholic fibrosing steatohepatitis, indicating that dysregulation of mi R-146a-5p might be critical in the pathogenesis of non-alcoholic fibrosing steatohepatitis.Part 2 Prediction of target genes and bioinformatic analysis of the differentially expressed mi RNAsObjective: To predict the target genes of dysregulated mi RNAs and bioinformatic analysis of the predicted target genes.Methods: Three bioinformatic resources Target Scan, Pic Tar and mi Randa were performed to predict the target genes of dysregulated mi RNAs.Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG)were then used to analyze the function and the signaling pathways of the predicted target genes. q RT-PCR and Western blot were pperformed to explore the expression levels of mi R-146a-4p target genes at the m RNA and protein levels. The validation of the identified binding sites on the mi R-146a-5p was conducted by dual-luciferase reporter gene assay.Results:1 Bioinformatic analysis of the predicted target genes of the differentially expressed mi RNAs: GO analysis revealed that the functions of predicted target genes were enriched mainly in transcription, transcription enzyme activity, protein binding and phosphorylation. KEGG analysis showed that the enriched pathways were mainly in the category of insulin signaling, apoptosis,Wnt signaling pathway, Toll-like receptor signaling pathway and m TOR signaling pathway.2 Prediction of mi R-146a-5p target genes: three bioinformatic resources Target Scan, Pic Tar and mi Randa were used to predict the target genes of mi R-146a-5p. Among the predicted targets of mi R-146a-5p, Wnt1 and Wnt5 a were predicted as potential target genes of mi R-146a-5p. Analysis of the homology showed that the 7 nucleotides in the seed region of mi R-146a-5p were complementary to bases of Wnt1 and Wnt5 a, respectively. No perfect binding site for mi R-146a-5p in the coding region of Wnt1 and Wnt5 a was identified.3 Results of dual-luciferase reporter gene assay: to determine whether mi R-146a-5p directly binds to the predicated sites on the 3’Untranslated Regions(3’UTR) of Wnt1 and Wnt5 a, we performed luciferase reporter assays. mi R-146a-5p mimics significantly reduced Wnt1 3’UTR-dependent and Wnt5 a 3’UTR-dependent luciferase activity but did not affect the luciferase activities of the mutant reporter, whereas mimic control had no effect on wild type or mutant reporter luciferase activity. On the other hand,mi R-146a-5p inhibitor enhanced wild type or mutant reporter luciferase activity. These results suggested an interaction between mi R-146a-5p and the3’UTR of Wnt1 and Wnt5 a m RNA.4 The expression of mi R-146a-5p target genes in mouse liver tissues: we further explored the impact of mi R-146a-5p on the expression levels of Wnt1 and Wnt5a. Down-regulation of hepatic mi R-146a-5p led to a significant up-regulation of the m RNA and protein expressions of Wnt1 and Wnt5 a in the livers of mice fed with MCD diet, as compared to the control mice.Conclusions:1 The differentially expressed mi RNAs may mediate the progression of non-alcoholic fibrosing steatohepatitis via regulating multiple signaling pathways.2 mi R-146a-5p may directly bind to the predicated sites of the Wnt1 and Wnt5 a 3’UTR, suggesting an interaction between mi R-146a-5p and the3’UTR of Wnt1 and Wnt5 a m RNA.Part 3 Effects and mechanisms of mi R-146a-5p on regulating hepatic stellate cellsObjective: To investigate the effects and mechanisms of mi R-146a-5p on the regulation of hepatic stellate cells(HSCs).Methods: Mouse primary HSCs were isolated by in situ digestion with pronase/collagenase, and purified by Percoll density gradient centrifugation.q RT-PCR and Western blot were performed to evaluate the hepatic expression of mi R-146a-5p and targe genes. HSC-T6 and LX-2 cells were transfected with 50 μM mi R-146a-5p mimics and 100 μM inhibitor. [50 μM and 100 μM?Why use such a high concentration?] Cell counting kit 8(CCK8) was used to measure cell ability, and q RT-PCR and Western blot were performed to evaluate the expression of mi R-146a-5p, target genes, and fibrogenics genes.HSC-T6 cells were transfected with si RNAs against Wnt1 and Wnt5 a, and q RT-PCR and Western blot were performed to evaluate the expression of mi R-146a-5p, target genes, and fibrogenics genes.Results:1 Expression of mi R-146a-5p and target genes during HSC activation: In HSCs cultured for 1-7 days, there was a gradual but significant reduction of mi R-146a-5p expression, whereas the expression of target genes Wnt1 and Wnt5 a were significantly increased.2 Effect of mi R-146a-5p overexpression on the proliferation of HSCs:CCK8 assay showed that overexpression of mi R-146a-5p by mimics significantly inhibited viability of HSC-T6 cells3 Effects of mi R-146a-5p modulation on HSCs activation and collagen deposition: Overexpression of mi R-146a-5p suppressed the m RNA and protein expression of α-smooth muscle actin(α-SMA), Collagen Type 1(Col-1), and matrix metalloproteinase-2(MMP-2), but increased the m RNA and protein expression of Smad7. On the contrary, the expression patterns of these genes at the m RNA and protein levels were reversed by mi R-146a-5p silencing, suggesting that mi R-146a-5p may suppress HSC activation and inhibit ECM deposition.4 mi R-146a-5p mimics significantly down-regulated the expression ofβ-catenin and NFAT5 but up-regulated the expression of GSK-3β both at the m RNA and protein levels. In contrast, silencing mi R-146a-5p led to opposite changes, indicating that mi R-146a-5p is involved in the regulation of Wnt signaling pathway.5 Knockdown of Wnt1 or Wnt5 a inhibited Wnt signaling and fibrogenesis: by q RT-PCR and Western blot, we have confirmed that Wnt1 and Wnt5a were significantly down-regulated after si RNA transfection.Knockdown of Wnt1 or Wnt5 a significantly reduced the expression ofβ-catenin and NFAT5 but elevated the expression of GSK-3β, both at the m RNA and protein levels. Moreover, knockdown of Wnt1 or Wnt5 a significantly suppressed the expression of the pro-fibrotic genes α-SMA,Col-1 and MMP-2, but enhanced the expression of Smad7.Conclusions:1 mi R-146a-5p may be involved in the pathogenesis of non-alcoholic fibrosing steatohepatitis by regulating the proliferation, activation and collagen deposition of HSCs.2 mi R-146a-5p may inhibit the pathogenic process of non-alcoholic fibrosing steatohepatitis by interfering Wnt1, Wnt5 a and Wnt signaling pathway.Part 4 Effect of heme oxygenase 1(HO-1) on the expression of mi R-146a-5p in mice with non-alcoholic fibrosing steatohepatitisObjective: To clarify the effect of HO-1 on the pathological process of non-alcoholic steatohepatitis in mice.Methods: C57BL/6J mice were fed with MCD diet for 8 weeks to induce steatohepatits. HO-1 inducer(Hemin) and inhibitor(Zinc protoporphyrin,Zn PP) were administrated to mice by intraperitoneal injection. Serum ALT and AST levels were measured by automatic biochemical analyzer. H&E and Masson staining were used to evaluate steatosis, inflammation, and liver fibrosis in liver sections. q RT-PCR and Western blot were performed to detect the expression of mi R-146a-5p, Wnt1 and Wnt5 a, as well as the downstream factors of Wnt signaling pathway and fibrogenic genes.Results:1 Serum ALT and AST changes: mice fed MCD diet showed significantly higher level of ALT and AST in serum. Hemin administration led to a significant reduction of serum ALT and AST(P<0.05, compared to controls).Conversely, mice treated with Zn PP showed much higher ALT and AST levels,as compared to MCD fed mice(P<0.05).2 Histology and pathology: liver sections from mice fed MCD diet for 8weeks showed disordered lobule structure, macrosteatosis in Zone 3, spot or focal hepatocyte necrosis, inflammatory infiltration and perisinusoidal fibrosis.After Hemin administration, hepatic steatosis, necrotic inflammation and progressive fibrosis were significantly ameliorated. However, Zn PP administration could notably aggravate liver injury, as compared to MCD fed mice.3 Effect of HO-1 on the expression of mi R-146a-5p and target genes:HO-1 inducer Hemin could significantly up-ragulate the expression of mi R-146a-5p, decrease the expression of target genes Wnt1, Wnt5 a and downstream factors β-catenin, NAFT5. Hemin also increases the expression of GSK-3β. Zn PP administration could down-regulate the expression of mi R-146a-5p, but increase the expression of Wnt1, Wnt5 a, β-catenin, and NAFT5, and decrease the expression of GSK-3β.Conclusions:1 HO-1 could ameliorate liver injury in non-alcoholic fibrosing steatohepatitis.2 HO-1 may play a protective role in non-alcoholic fibrosing steatohepatitis through modulating mi R-146a-5p and target genes Wnt1,Wnt5 a.