| Non-alcoholic fatty liver disease(NAFLD)is the most common liver disease in developed countries.The global prevalence rate of about 25%,China is also up to 15%.NAFLD is becoming one of the public health concerns of the world.NAFLD is associated with insulin resistance and metabolic syndrome.The treatment of type 2 diabetes mellitus for insulin resistance has some effect on the treatment of NAFLD,such as controlling weight loss,metformin and thiazolidinediones.Up to 80% of obese suffer from this disease.Non-alcoholic steatohepatitis(NASH)is a severe pathologic process of NAFLD,accounting for 15%-25% of all NAFLD patients,and is considered to be the leading cause of cirrhosis.miRNAs are endogenous,non-encoded non-coding small RNA fragments of about 19-25 nucleotides.It can recognize the 3'-untranslated region(3'-UTR)specific binding site of its target m RNA,depending on the individual sequence,leading to repression or m RNA degradation.miRNA plays a significant role in cell proliferation,development,differentiation,and apoptosis.miRNAs also play a significant role in the development of many chronic liver diseases,including viral hepatitis,drug-induced liver injury and autoimmune liver disease.In this study,we established the NASH-related liver fibrosis mouse model by MCD diet and applied the m RNA microarray,bioinformatics analysis,dual luciferase reporter gene system,hepatic stellate cell culture,real-time quantitative PCR(QRT-PCR),Western blotting,MTT,flow cytometry,si RNA transfection and rescue experiment,etc.Hepatic stellate cell experiment directly controlled the effect and mechanism of miR-130a-3p on the hepatic fibrosis of NASH,from the activation,proliferation,apoptosis and activation and collagen deposition of stellate cells.The results of this study contribute to an in-depth understanding of the pathogenesis of hepatic fibrosis and provide new potential targets for the treatment of NAFLD.Part 1 Screening and validation of differentially expressed miRNAs in liver tissue after NASH-associated liver fibrosis in mouse MCD modelObjective: To establish a non-alcoholic fibrosing steatohepatitis animal model by MCD diet,screen and validate the expression profiles of dysregulated miRNAs in the liver tissue.Methods: Eight weeks old male C57 BL / 6J mice were fed with methionine-choline deficient(MCD)diet to construct NASH-related liver fibrosis mouse model.To evaluate the changes of serum biochemical and histopathological changes in mice,we analyzed the miRNAs by LC Sciences micro RNA Microarray-20.0 Mouse miRNA gene expression profiling.Statistics screened the miRNAs and verified by QRT-PCR.Results:1 The changes of serum ALT and AST in mice with NASH-related hepatic fibrosis were significantly higher than those in control group(P < 0.05).2 Liver histopathology: MCD model NASH-related liver fibrosis in mice liver tissue showed hepatic steatosis,mainly to the massive bubble,with focal necrosis and inflammatory cell infiltration,sinus fibrous tissue hyperplasia.3 Differentially expressed miRNAs of liver tissue in mice with hepatic fibrosis of NASH: There were 37 differentially expressed miRNAs in liver tissue of NASH-related liver fibrosis mice(P < 0.05).The miRNAs of miR-582-3p,miR-210-3p,miR-211-5p,miR-7215-5p and miR-216a-5p were significantly up-regulated,while miR-335-5p,miR-130a-3p,miR-7236-3p,miR-146a-5p and miR-30a-5p were significantly down-regulated in the liver tissue of the model group.4 Differential expression of miRNAs: QRT-PCR results showed that the expression of miR-582-3p was significantly up-regulated compared with Control group,and the expression of miR-130a-3p and miR-335-5p was significantly down-regulated(P < 0.05),which results were consistent with the microarray.Part 2 Bioinformatics Analysis of Differential Expression of miRNAs and Selection and Verification of Target GenesObjective: To predict the target genes of dysregulated miRNAs and bioinformatics analyze the predicted target genes.Then validate target genes with dual fluorescence and pathological results.Methods: The bioinformatics method was used to analyze GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Gene and Genomes)signaling pathways.And target gene prediction,combined with the pathogenesis of NASH-related path to determine the target gene.The specific binding of miR-130a-3p to target genes TGFBR1 and TGFBR2 was confirmed by double luciferase reporter gene system.In situ hybridization,immunohistochemistry and m RNA and protein validation of target genes and related factors were performed in mouse model tissues.And the results were also confirmed in the organization of NASH patients.Results:1 Differential expression miRNAs target gene prediction and bioinformatics analysis: A total of 19069 potential target genes were identified,of which 671 genes were associated with down-regulated miRNAs,920 genes were associated with up-regulated miRNAs,and 17478 genes were related both to down-regulated and up-regulated miRNAs.The expression of miRNAs was significantly enriched in the cytoskeleton,ion binding,RNA regulatory metabolic processes,Wnt signaling pathway and TGF-? / SMAD signaling pathway,apoptotic pathway and ECM-receptor interaction signaling pathway.Upregulation of differentially expressed genes of miRNAs was significantly enriched in mitochondria,DNA binding,transcription,Wnt signaling and MAPK signaling,insulin signaling,ECM-receptor interactions and apoptotic pathways.2 Mi R-130a-3p and target gene specificity: The target genes of miR-130a-3p were predicted by five miRNA target prediction programs(Targetscan,miRanda,miRDB,PITA and RNA22),in which the binding sites of miR-130a-3p were present in both TGFBR1 3'-UTR and TGFBR2 3'-UTR.The results showed that the 3'-UTR wild-type plasmids of TGFBR1 / 2 genes were co-transfected with miR-130a-3pmimics compared with control group.Dual luciferase reporter gene verified the binding relationship between the target gene and miR-130a-3p.The results showed that: Compared with control group,the 3'-UTR wild-type plasmid of TGFBR1 / 2 genes was co-transfected with miR-130a-3pmimics to significantly reduce the luciferase activity of the reporter gene(P < 0.05).miR-130a-3p inhibitor,the luciferase activity of the reporter gene was significantly increased(P < 0.05).The miR-130a-3p mimics,mimics control,miR-130a-3p inhibitor,inhibitor control had no effect on the activity of 3'-UTR mutant plasmid luciferase in TGFBR1 and TGFBR2 gene(P > 0.05).3 Verification of Mi R-130a-3p and target genes and related factors in liver tissue: 3.1)Validation in mouse tissue: 3.1.1)The expression of m RNA and protein of liver tissue fibrosis related genes TGFBR1,TGFBR1,smad2,smad3,Col-1 and Col-4 in MCD group was significantly higher than that in control group(P < 0.05).3.1.2)Liver tissue sections were compared with the control group: In situ hybridization results suggest that miR-130a-3p expression was significantly reduced,while Immunohistochemical results indicate that expression of TGFBR1,TGFBR1,Col-1 and Col-4 was significantly increased.3.2)Confirmation in patients with tissue sections: NASH patients with liver fibrosis with liver tissue showed hepatic steatosis,mainly to the big bubble,with focal necrosis and inflammatory cell infiltration,sinus fibrous tissue hyperplasia.Compared with the control group,the In situ hybridization results showed that the expression of miR-130a-3p was significantly decreased in the liver tissue of patients with hepatic fibrosis in NASH patients.The results of immunohistochemistry showed that expression of TGFBR1,TGFBR2,Col-1 and Col-4 was significantly increased.Part 3 The effects and mechanisms of miR-130a-3p on the function of hepatic stellate cells in NASH-related hepatic fibrosisObjective: To investigate the effects and mechanisms of miR-130a-3p on the role of hepatic stellate cells(HSCs)in NASH-related hepatic fibrosis.Methods: The expression of miR-130a-3p and target gene at 0d,1d,3d and 7d were detected by HSC-T6 in vitro.At the same time,miR-130a-3p mimics were transfected into hepatic stellate cell line overexpression,MTT assay and flow cytometry(Annexin V / PI)and related apoptosis factor were used to detect apoptosis rate.RT-PCR and Western blot analysis were used to analyze the expression of target genes,downstream signaling molecules,and hepatic fibrosis related factors.To investigate the mechanism of miR-130a-3p by acting on target genes TGFBR1 and TGFBR2 on fatty liver fibrosis.Results:1 Changes of miR-130a-3p and target gene expression during hepatic activation of hepatic stellate cells(0d,1d,3d and 7d): 1.1)miR-130a-3p expression,1d vs.0d,7d vs.3d were significantly decreased(P < 0.05).1.2)expression of m RNA: TGF-?1: 1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).?-SMA,3d vs.1d was significantly increased(P < 0.05).Smad2,7d vs.3d was significantly increased(P < 0.05).Smad2,7d vs.3d increased was significantly(P < 0.05).Expression of protein: TGF-?1: 1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).?-SMA,1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).Smad2,1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).Smad3,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).1.3)expression of m RNA: TGFBR1,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).TGFBR2,3d vs.1d was significantly increased(P < 0.05).Expression of protein: TGFBR1,1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).TGFBR2,1d vs.0d,3d vs.1d,7d vs.3d were significantly increased(P < 0.05).2 Overexpression of miR-130a-3p in hepatic stellate cells [Mi R-130a-3p mimics(M)group,Control(C)group and mimics control(MC)group]: 2.1)miR-130a-3p m RNA levels in hepatic stellate cells: M vs.C,M vs.MC were significantly increased(P < 0.05).48 h vs.0 h were significantly increased after transfection(P < 0.05).2.2)Proliferation rate: M vs.C,M vs.MC were significantly decreased at 48 h and 72h(P < 0.05).2.3)Apoptosis of hepatic stellate cells: 2.3.1)Apoptotic rate: M vs.C,M vs.MC were significantly increased(P < 0.05).2.3.2)Apoptosis-related factors: expression of m RNA: Caspase-3,Cleaved Caspase-9 and Cleaved PARP-1,M vs.C,M vs.MC were significantly increased(P < 0.05).Expression of protein: Cleaved Caspase-3,Cleaved Caspase-9 and Cleaved PARP-1,M vs.C,M vs.MC were significantly increased(P < 0.05).2.4)Effects of miR-130a-3p overexpression on activation of hepatic stellate cells and collagen deposition: expression of m RNA and protein: TGF-?1,Smad2,Smad3,Col-1,Col-4,MMP-2 and MMP-9,M vs.C,M vs.MC were significantly increased(P < 0.05).Part 4 Knockdown and Functional Recovery of TGFR1 and TGFBR2 of miR-130a-3p Target GeneObjective: To confirm the critical role of TGFBR1 and TGFBR2 in hepatic fibrosis and identified TGFR1 and TGFBR2 as the primary target of miR-130a-3p in NASH-related hepatic fibrosis.Methods: The HSC were cultured in vitro,and si RNA transfection silenced TGFBR1 and TGFBR2.The miR-130a-3p mimics were co-transfected with TGFBR1 and TGFBR2 wild-type plasmids to the hepatic stellate cell line for target protein recovery.The changes of TGFBRs,TGF-? / SMAD downstream signal molecules,and hepatic fibrosis related factors were detected in HSC activation.Logically confirmed that miR-130a-3p regulates NASH by acting on its target genes TGFBR1 and TGFBR2.Results:1 Effects of si RNA silencing TGFBR1 and TGFBR2 on TGF-? / SMAD downstream signaling molecules and hepatic fibrosis related factors[Control(C)group,si-Control(SC)group,si-TGFBR1(S1)group,si-TGFBR2(S2)group and(si-TGFBR1 + si-TGFBR2)(SA)group]:1.1)Detection of silencing efficiency of si TGFBR1 and si TGFBR2: expression of m RNA and protein :TGFBR1,S1 vs.C,SA vs.C were significantly decreased(P < 0.05).TGFBR2,S2 vs.C,SA vs.C were significantly decreased(P < 0.05).1.2)Effects of si RNA silencing TGFBR1 and TGFBR2 on downstream signaling molecules of TGF-? / SMAD: expression of m RNA and protein: TGF-?1,S1 vs.C,S2 vs.C,SA vs.C were not statistically significant(P > 0.05).?-SMA,Smad2,and Smad3,S1 vs.C,S2 vs.C,SA vs.C,S1 vs.SC,S2 vs.SC,SA vs.SC were significantly decreased(P < 0.05).1.3)Effect of si RNA silencing TGFBR1 and TGFBR2 on hepatic fibrosis related factors: expression of m RNA and protein: MMP-2,MMP-9,Col-1 and Col-4,S1 vs.C,S2 vs.C,SA vs.C,S1 vs.SC,S2 vs.SC,SA vs.SC were significantly decreased(P < 0.05).2 Results of miR-130a-3p mimics co-transfection with TGFBR1 and TGFBR2 plasmids for rescue experiment [ Control(C)group,mimics-Control(MC)group,miR-130a-3p mimics(M)group,(miR-130a-3p mimics + TGFBR1)(M1)group,(miR-130a-3p mimics + TGFBR2)(M2)group and(miR-130a-3p mimics + TGFBR1 + TGFBR2)(MA)group] : 2.1)On TGFBR1 and TGFBR2: expression of m RNA: TGFBR1,M vs.C was significantly decreased(P < 0.05).MA vs.M was significantly increased(P < 0.05).TGFBR2,M vs.C was significantly decreased(P < 0.05).M2 vs.M,MA vs.M were significantly increased(P < 0.05).Expression of protein: TGFBR1,M vs.C was significantly decreased(P < 0.05).M1 vs.M,MA vs.M was significantly increased(P < 0.05).TGFBR2,M vs.C was significantly decreased(P < 0.05).M2 vs.M,MA vs.M were significantly increased(P < 0.05).2.2)On TGF-? / SMAD downstream signaling molecules and hepatic fibrosis related factors: expression of m RNA: TGF-?1,M vs.C was not statistically significant(P > 0.05).Smad2,M vs.C was significantly decreased(P < 0.05).MA vs.M was significantly increased(P < 0.05).Smad3,M vs.C was significantly decreased(P < 0.05).MA vs.M was significantly increased(P < 0.05).Col-1,M vs.C was significantly decreased(P < 0.05).MA vs.M was significantly increased(P < 0.05).Col-4,M vs.C was significantly decreased(P < 0.05).MA vs.M was significantly increased(P < 0.05).Expression of protein: TGF-?1,M vs.M1,M vs.M2,M vs.MA were significantly decreased(P < 0.05).Smad2,M vs.C was significantly decreased(P < 0.05).M2 vs.M,MA vs.M were significantly increased(P < 0.05).Smad3,M vs.C was significantly decreased(P < 0.05).M2 vs.M,MA vs.M were significantly increased(P < 0.05).Col-1,M vs.C was significantly decreased(P < 0.05).M1 vs.M,M2 vs.M,MA vs.M were significantly increased(P < 0.05).Col-4,M vs.C was significantly decreased(P < 0.05).M1 vs.M,M2 vs.M,MA vs.M were significantly increased(P < 0.05).Conclusion:1 MCD diet can successfully establish C57 BL / 6J mouse NASH-related liver fibrosis animal model.It is a good guarantee to study the pathogenesis of the disease thoroughly.2 Mi RNAs in mouse liver tissue changed significantly during NASH-related hepatic fibrosis.Differentially expressed miRNAs may be involved in the development of NASH-associated liver fibrosis and participate in the regulation of pathologic mechanisms.3 Mi R-130a-3p expression in NASH-related hepatic fibrosis and hepatic stellate cell activation is significantly reduced,which can inhibit the proliferation,activation and collagen deposition of HSC,while inducing apoptosis,thereby delaying or preventing the progress of liver fibrosis.4 Knockdown of TGFBRs and Rescue results suggest that miR-130a-3p may play a significant role in the development of NASH-associated hepatic fibrosis by specifically inhibiting target genes TGFBR1 and TGFBR2. |
PDF Full Text Request