Screening And Function Study Of Non-alcoholic Fibrosing Steatohepatitis Related MicroRNAs

Nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of themetabolic syndrome, has similar pathological changes with alcoholic liverdisease, but the patients suffer from it without excessive alcohol intake andother definite etiological factor. It has been reported that NAFLD is relatedwith insulin resistance and hereditary susceptibility. The disease spectrum ofNAFLD includes simple fatty liver (SFL) non-alcoholic steatohepatitis(NASH), NASH-related hepatic fibrosis and cirrhosis, hepatocellularcarcinoma. NASH is the key stage in the spectrum of NAFLD which ischaracterized by non-alcoholic steatohepatitis／fibrosing steatohepatitis andcan progress to fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma.With the changes of life style and diet, the prevalence of NAFLDincreases rapidly in modern society. NAFLD has became the most commonchronic liver diseases in the United States and worldwide. Recent studiesusing different methodologies indicate that in the general population theprevalence of NAFLD is approximately20%~40%. Of greater concern is therecognition that cirrhosis and liver-related deaths occur in approximately20%and8%of these NASH patients over a10-year period. Among more affluentregions of China, the community prevalence of non-alcoholic fatty liverdisease (NAFLD) is approximately15%. With the increasing pandemic ofobesity, the prevalence of NAFLD has approximately doubled in the pastdecade. Nonalcoholic steatohepatitis/fibrosis, one type of a larger spectrum ofnonalcoholic fatty liver disease, is now recognized as a cause of progressivefibrotic liver disease with adverse clinical sequelae. However, thepathogenesis of nonalcoholic steatohepatitis/fibrosis leading to diseaseprogression remains poorly understood and definitive therapies for thesepatients are lacking． MicroRNAs (miRNAs) are a class of important factors in the regulation ofprotein expression at the posttranscriptional level. miRNAs are associatedwith many pathophysiologic events such as hepatic tissue development anddifferentiation, cell proliferation, apoptosis. In the study of Sanyal and hiscolleagues, they found that NASH was associated with altered hepaticmiRNAs expression including the upregulation of miR-34a and miR-146b,downregulation of miR-122. miR-122has been found to be required for thelipid homeostasis through the regulation of sterol-regulatory element-bindingprotein (SREBP), fatty acid synthetase (FAS) and HMG CoA reductase(HMGCR). miR-33a and miR-33b have a crucial role in controllingcholesterol and lipid metabolism in concert with their host genes, the SREBPtranscription factors. miR-34family has been found to control cholesterolsynthesis by targeting acyl-CoA synthetase long-chain family member1(ACSL1) and inhibiting sirtuin-1with downstream dephosphorylation ofAMP kinase and HMGCR. Downregulation of miR-467b is involved in thedevelopment of hepatic steatosis by modulating the expression of its targethepatic lipoprotein lipase (LPL). miR-103and miR-107regulate insulin andglucose homeostasis through insulin receptor CAV1. Dysregulation ofmiRNAs may contribute to the development of NAFLD, however, the exactroles of miRNAs in the pathogenesis of NASH are not fully understood.In this study， we aimed to establish a non-alcoholic fibrosingsteatohepatitis model caused by methionine and choline deficient (MCD) diet8weeks. Serum alanine aminotransferase (ALT) and aspartateaminotransferase (AST), reliable indicators of liver inflammatory injury, weremeasured. Histological scoring for NAFLD was carried out to evaluate hepaticsteatosis and fibrosis. Using microarray experiments, we found that feedingC57BL/6J mice a methyl-deficient diet8weeks to induce non-alcoholicfibrosing steatohepatitis could result in aberrant expression of miRNAs. Toclarify the roles of miRNAs in the process of non-alcoholic fibrosingsteatohepatitis and its molecular mechanism, and provided a basis for NAFLDprevention and the development of miRNA targeting drugs, the different expressed miRNAs were performed with the bioinformatic analysis. Thederegulated miRNA and miRNA targeting gene was validated usingquantitative real-time PCR and western blot assay.To study the role of these miRNAs in the NASH, levels of miR-122,miR-199a-5p and miR-221were determined in the livers extracted from micefed MCD and control diets, using quantitative real-time PCR experiments.To investigate the potential interaction experimentally, the wild-type3′-UTRof NCOR1gene was subcloned downstream of the Renilla luciferase codingsequence and cotransfected into293T cells with the pre-miR-199a-5p andnegative control sequence. The luciferase activity was assayed at48hpost-transfection. To examine the effect of miR-199a-5p, we transfected itsmimic, inhibitor and Ncontrol (NC) into a hepatic stellate cellline named LX-2cells and observed the expression of several fibrosis-associated genes,providing new insights to the pathogenesis of NASH.Part1：Alterations and validation of microRNA expression profile innon-alcoholic fibrosing steatohepatitis modelObjective: To establish a non-alcoholic fibrosing steatohepatitis modeland study the expression profiles of fibrosis related miRNAs.Methods: Experimental non-alcoholic fibrosing steatohepatitis modelswere established by feeding mice with MCD diet8weeks (MCD group).Control mice were fed with choline–methionine supplemented diet (controlgroup). Serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were measured by enzymic method with automatic biochemistryanalyzer. Hepatic inflammation and fibrosis were graded under HE andMasson staining. The stage of steatosis and fbrosis was assessed according tothe guidelines for diagnosis and treatment of non-alcoholic fatty liver diseases.RNAs from experimental mice hepatic tissue were isolated with a MiRNeasyMini Kit (Qiagen). The miRNAs profiles were determined using GeneChipmiRNA2.0Array (Affymetrix). The result of microarray was validated byreal-time quantitative PCR. Results:1. General observation: The mice of MCD group showed less activity,sikness and dim hair than the mice of Control group.2. Liver index changes: The body weights of MCD group mice weresignificantly lower than the control group at the8th week body (P <0.05).Liver wet weight of MCD group mice had no significant change comparedwith the control group (P>0.05). Liver index (liver weight/body weight×100%) was significantly higher than the control group (P <0.05).3. Serum transaminases in C57BL/6J mice fed control and MCD diet for8weeks: MCD diet increased the level of serum transaminases, both ALT andAST.4. HE and Masson staining of liver sections of C57BL/6J mice fedcontrol and MCD diets: MCD treated animals showed disordered lobulestructure, severe macrosteatosis, focal hepatocyte necrosis, inflammatoryinfiltration, portal fibrosis and fibrous septum.5. The result of GeneChip miRNA2.0Array showed that there were47deregulated miRNAs in mice hepatic tissue treated with MCD or control diet,in which15miRNAs changed above2times. The result of microarray showedthat10miRNAs were upregulated in MCD group including mmu-let-7i,mmu-mir-155, mmu-mir-199a-5p, mmu-mir-34a, mmu-mir-221,mmu-mir-200c, mmu-mir-297c, mmu-mir-713, mmu-mir-190b, mmu-mir-678;5miRNAs downregulated including mmu-mir-122, mmu-mir-103,mmu-mir-146, mmu-mir-101a and mmu-mir-466j.6. Refer to the relevant literature and the results of microarray, weselected miR-122, miR-199a-5p and miR-221to perform quantitativereal-time PCR experiments. We found that MCD diet increased miR-199a-5pand decreased miR-122in mice livers (P <0.05).Conclusion:1. MCD diet could induce characteristic pathology of steatosis, mixedhepatic inflammatory and fibrosis, representing a successful model ofnon-alcoholic fibrosing steatohepatitis.2. Non-alcoholic fibrosing steatohepatitis in mice significantly change the expression of miRNA profiles, which may be involved in non-alcoholicsteatohepatitis/fibrosing steatohepatitis.3. We found a dramatic upregulation of miR-199a-5p in the non-alcoholicfibrosing steatohepatitis mouse model. These data indicated that dysregulatedexpression of miR-199a-5p might be critical in non-alcoholic fibrosingsteatohepatitis pathogenesis.Part2：Functional analysis and evaluation of miR-199a-5p target genesObjective: To searche the target genes of miR-199a-5p and detect therole of miR-199a-5p in non-alcoholic fibrosing steatohepatitis etiology.Methods: The DAVID (Database for Annotation, Visualization andInterrogated Discovery) bioinformatic resources were performed to analysisthe function of miR-199a-5p. Three most commonly prediction algorithmsTargetScan6.2, miRanda and PITA was used to find the exact target genes ofmiR-199a-5p. Target gene of miR-199a-5p in the experimental mice hepatictissue was evaluated by real-time quantitative PCR and western blot assay.Results:1. Enrichment analysis on predicted target genes: Result obtained fromGO-term analysis revealed a relationship between miR-199a-5p and regulationof transcription, transcription factor complex, protein serine/threonine kinaseactivity, and others. KEGG analysis showed the involvement of miR-199a-5pin a variety of pathways, including insulin signaling, Wnt signaling andMAPK signaling pathways.2. Prediction of miR-199a-5p target genes:26target genes ofmiR-199a-5p were predicted by all of the three algorithms. NCOR1(nuclearreceptor corepressor1), one of the predicted target genes, was reported to berequired for the integration of inflammatory and anti-inflammatory signals,which is associated with the pathogenesis of non-alcoholic fibrosingsteatohepatitis.3. Expression of NCOR1in hepatic tissues: Increased hepaticmiR-199a-5p upon MCD diet is associated with decreased NCOR1expressionat the protein, but not mRNA level. Conclusion:1. Results of bioinformatic analysis showed that miR-199a-5p may takepart in the pathogenesis of non-alcoholic fibrosing steatohepatitis throughinsulin signaling, Wnt signaling and MAPK signaling pathways.2. miR-199a-5p may regulate the translation of NCOR1mRNA.Part3：The interaction between miR-199a-5p and its target gene NCOR1Objective: To investigate the potential interaction between miR-199a-5pand its target gene NCOR1mRNA3′-UTR experimentally.Methods: The3′-UTR of NCOR1was PCR amplified and cloneddownstream of the renilla luciferase stop codon in psiCHECKTM-2vector,giving rise to NCOR13′-UTR-luciferase. This construct luciferase reportervectors were transfected into HEK293cells with pre-miR-199a-5p ornegative control. Cells were harvested48h after transfection and assayed withDual Luciferase Reporter Assay System according to the manufacturer’sinstructions.Results:1. From “Ensembl Mouse database”, we obtained a full length2202bpNCOR13’-UTR sequences, where may be binding sites with miR-199a-5p inNCOR13’-UTR of790bp~796bp place.2. Genomic DNA was extracted from the blood and amplified by PCRassay. The PCR products showed a specific treaty2000bp fragment on a1.0%agarose gel electrophoresis which was in line with expectations. Therecombinant plasmid p3’UTR-NCOR1was confirmed by Shanghai Sangonfurther sequencing of plasmid.3. The luciferase activity assays at48h post-transfection showed that theoverexpression of miR-199a-5p could decrease relative luciferase activitysignificantly.Conclusion: miR-199a-5p may play important roles in the pathogenesisof non-alcoholic fibrosing steatohepatitis by influencing protein translation ofNCOR1. Part4The regulation of NCOR1and fibrosis-related genes bymiR-199a-5p was examined by silencing or overexpressing this miRNA inLX-2cellsObjective: To observe the expression of several fibrosis-associated genesby regulating the expression of miR-199a-5p in LX-2cells, further detect therole of miR-199a-5p in the pathogenesis of non-alcoholic fibrosingsteatohepatitis and prevention and provide a new target basis to treatnon-alcoholic fibrosing steatohepatitis.Methods: Human hepatic stellate cells (LX-2cells) were cultured inDMEM supplemented with10%fetal bovine serum,100U/mL penicillin, and100g/L streptomycin at37°C in a humidified environment with5%CO2.Cells were grown to60%confluency prior to transfection with differentconcentrations of miR-199a-5p mimic, inhibitor, or their correspondingcontrols using Lipofectamine2000. Cell viability was determined by MTTassay in each group to select the appropriate concentration of transfection.Transfected cells were cultured for48hours and harvested for total RNA andprotein extraction. The samples were stored at80°C for q-PCR or Westernblot assays.Results:1. The result of MTT assay showed that transfection with controlsequences of different concentrations without significant effect on cellviability; transfection of100nM and150nM miR-199a-5p mimic, cell viabilitywas significantly reduced, but no significant changes with50nM mimic;transfection of100nM and150nM miR-199a-5p inhibitor, cell viability wassignificantly enhanced, but no significant changes with50nM inhibitor.2. Compared with the control group, the expression of miR-199a-5pdramatically increased in mimic group and significantly reduced in inhibitorgroup. NCOR1mRNA was not affected by miR-199a-5p mimic, inhibitor andNcontrol (NC).3. NCOR1protein was decreased by miR-199a-5p overexpression andincreased by miR-199a-5p knockdown (p <0.05) in LX-2cells, while PPAR γprotein expressed inversely of NCOR1protein in each group. 4. miR-199a-5p inversely correlated with the expression of severalfibrosis-associated genes, including transforming growth factor-β (TGF-β) andα-smooth muscle actin (α-SMA).Conclusion:1. miR-199a-5p may influence cell proliferation or apoptosis involved inthe pathogenesis of non-alcoholic fibrosing steatohepatitis.2. miR-199a-5p may affect PPAR γ involved in the activation of hepaticstellate cells through negative regulation the expression of NCOR1.3. Targeted regulation of miR-199a-5p expression may prevent or reversethe occurrence and development of hepatic fibrosis.