MiR-484 Promote Hepatic Fibrosis Through Targeting HIPK1

BackgroudLiver fibrosis is a pathological process that can be caused by all kinds of harmful factors,and the deposition of extracellular matrix(ECM)can severely damage the liver’s normal structure and function.Liver fibrosis is the common pathological process of various chronic liver diseases,and it can develop into liver cirrhosis even liver cancer after 20~40 years’ no symptoms state.Therefore,the early diagnosis and treatment of liver fibrosis become a top priority.Until now,the liver biopsy is still the gold standard for the diagnosis of liver fibrosis.However,it not be widely used because it’s a invasive measurement,the non-invasive strategy against liver fibrosis still need to be found,implying that it’s necessary to do further research on the molecular mechanism involved in the development of this disease.The micro RNAs(miRNAs)are noncoding RNAs of about 20~25 nucleotides that usually negatively modulate the expression of target gene,as we know,it was wildly popular in various research filds such as endocrinology,cardiology and oncology.Existing studies have shown that the miRNAs also closely related to liver fibrosis by influencing HSC(hepatic stellate cell)cellular processes,including proliferation,apoptosis and migration.Our group engaged in the study of miRNAs on the occurrence and development of liver fibrosis for many years,and the previous work found that miR-484 was differently expressed in DMN induced rat liver fibrosis model(the article has been published).In order to further investigate whether miR-484 can affect HSC cell func-tion,we extracted the rat primary HSC and detected the expression changs of miR-484 during the cells spontaneously activation.We further found that HIPK1 is the direct target of miR-484 by bioinformatics analysis and dual-luciferase reporter assay system experiment.Existing research shows that HIPK1 play an important role in diverse biological processes such as cell proliferation,activation and apoptosis.This study found that miR-484 can promote HSC activation and inhibit it’s apoptosis by targeting HIPK1,and Wnt/β-catenin signaling pathway was involved in this process.The results provide a new molecular biomarker for the diagnosis and treatment of liver fibrosis.ObjectiveThis study based on the previous works,and aimd to further explore the role of miR-484 in the development of liver fibrosis,HSC-T6 in vitro experiments was performed.The current study contains four sections:(1)Detection of miR-484 expression changs during rat primary HSC spontaneously activation;(2)Selection and validation of miR-484 target gene;(3)Exploration of miR-484 influnce on the activation and apoptosis of HSC-T6,and the signaling pathways involved in these processes.Methods1 Detection of miR-484 expression changs during rat primary HSC spontaneously activation(1)Extracted the rat primary HSCs,the purity of the cells were detected by autofluorescence;cells were quiescent during the first 2 days and activated after 14 days,the cells state were identified by immunofluorescence;(2)qRT-PCR were uesd for detecting the expression levels of miR-484 in different periods.2 Selection and validation of miR-484 target gene(1)Selected miR-484 as keyword,and searched the target genes in micro RNA.org,miRbase and Target Scan.Gene ontology(GO)analysis was performed to explore the biological functions it may participate in;(2)Dual-luciferase reporter assay system experiment was performed to verify that HIPK1 is the direct target gene of miR-484;(3)Transfection experiment was performed in HSC-T6 cell lines,miR-484 inhibitor /mimics/ negative control were transfected into the cells,total protein were extracted 48 h after transfection.Western Blot were used for detecting the expression changs of HIPK1 in different groups.3 Exploration of miR-484 influnce on the activation and apoptosis of HSC-T6,and the signaling pathways involved in these processes(1)After transfection experiment,qRT-PCR and Western Blot were used for detecting correspondingly exprssion changes of α-SMA and col?;(2)qRT-PCR and Western Blot were performed to detect the changes of Wnt-3a,Wnt-5a and β-catenin which related with Wnt/β-catenin signaling pathway;(3)Annexin V-FITC/PI double staining was performed for detecting apoptosis rate of the HSC-T6 in different transfetion groups.Results1 Detection of miR-484 expression changs during rat primary HSC spontaneously activation(1)Autofluorescence results indicate that the purity of primary HSCs can reach more than 90%,the cells within 2 days are oval and contains high lipid content,under 328 nm UV irradiation,the quiescent HSCs showed yellow-green intrinsic autofluorescence;after 14 days,the cells were completely activated and extent into stellate shape;immunofluorescence experiment show that α-SMA were significantly up-regulated in activated primary HSCs;(2)qRT-PCR results suggested the expression of miR-484 was increased significant ly during the primary HSCs spontaneously activation(P<0.05).2 Selection and validation of miR-484 target gene(1)The bioinformatics analysis results suggest that HIPK1 is the target genes of miR-484;(2)Dual-luciferase reporter assay verified that HIPK1 is the direct target gene of miR-484;(3)After transfection with miR-484 inhibitor in HSC-T6 cell lines,HIPK1 was significantly up-regulated on protein levels;HIPK1 was significantly down-regulated on protein levels when miR-484 mimics was transfected;3 Exploration of miR-484 influnce on the activation and apoptosis of HSC-T6,and the signaling pathways involved in these processes(1)The result of qRT-PCR and Western Blot indicated that,after transfection with miR-484 inhibitor,a-SMA and col? were down-regulated compared with control group(P<0.05);and there has no difference between blank and control group(P>0.05);(2)After transfetion experiment,qRT-PCR and Western Blot results suggested that the expression of Wnt-3a,Wnt-5a and β-catenin were decreased(P<0.05);(3)The results of Annexin V-FITC/PI double-staining indicated that the rate of apoptosis were rose significantly when miR-484 were down-regulated.Conclusions1 The expression of miR-484 was increased significantly during the primary HSCs spontaneously activation;2 HIPK1 is the direct target gene of miR-484;3 miR-484 can accelerate HSC activation and inhibit its apoptosis by targeting HIPK1,promote the synthesis of ECM and the progress of liver fibrosis;4 miR-484 activated Wnt/β-catenin signaling pathway when promote liver fibrosis.