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This study was designed to investigate the potential role of microRNA-29c (miR-29c) in biliary atresia-related fibrosis. The expression of miR-29c was determined in 15 pairs of peripheral blood samples from infants with biliary atresia (BA) and infants with non-BA neonatal cholestasis using quantitative real-time PCR. EMT was established by induction with TGF-β1 in HIBEpiC cells. MiR-29c was inhibited by lipofectamine transfection. The expressions of proteins related to epithelial–mesenchymal transition (EMT), i.e., E-cadherin, N-cadherin and vimentin, were determined using quantitative real-time PCR and western blotting. Direct interaction between miR-29c and DNMT3A and DNMT3B was identified using a luciferase reporter assay. The expressions of DNMT3A and DNMT3B were suppressed by treatment with SGI-1027. Patients with BA showed significantly lower miR-29c levels in peripheral blood samples than the control subjects. In vitro, TGF-β1-induced EMT significantly decreased the expression of miR-29c. Downregulation of miR-29c had a promotional effect on BA-related fibrosis in HIBEpiC cells, as confirmed by the decrease in E-cadherin and increase in N-cadherin and vimentin levels. MiR-29c was found to target the 3’UTR of DNMT3A and DNMT3B and inhibit their expression. Suppression of DNMT3A and DNMT3B reversed the effects of miR-29c downregulation on BA-related fibrosis in HIBEpiC cells. These data suggest that BA-related fibrosis is closely associated with the occurrence of EMT in HIBEpiC cells. MiR-29c might be a candidate for alleviating BA-related fibrosis by targeting DNMT3A and DNMT3B.

Background and objectives Biliary atresia (BA) is a pediatric liver disease characterized by fibro-obliteration and obstruction of the extrahepatic biliary system, that invariably leads to cirrhosis and even death, if left untreated for extended time. However, its pathology and etiology still remained unknown. In this study, we tested the expression of adducin 3 (ADD3), the gene identified as a susceptibility gene in BA by GWAS, and uncovered its upstream regulatory microRNA in the pathogenesis of BA. Methods In this study, 14 infants with BA and 14 infants with choledochal cyst (CC) were enrolled as experimental group and control group, respectively. ADD3 and microRNA-145 (miR-145) expression profiles in liver tissues of BA and CC were determined using qPCR. Luciferase reporter assay was performed to verify the direct interaction between miR-145-5p and ADD3 3’ Untranslated Regions (3’UTR). The Lentiviral vectors containing miR-145, miR-145-3p inhibitor, miR-145-5p inhibitor, empty vector were transfected into human hepatic stellate cell line (LX-2) to determine the functional effect of miR-145 on ADD3 expression at both mRNA and protein level. Results MiR-145 was shown to be down-regulated in liver tissues of infants with BA compared to CC (p = 0.0267). ADD3, verified as a target of miR-145-5p, was shown to be overexpressed in infants with BA at the mRNA level (p = 0.0118). Transfection of lentiviruses containing miR-145 into LX-2 cells decreased the expression of ADD3 at both mRNA and protein level compared to negative control group, and suppressed the expression of p-Akt at protein level. Conclusions Our study has shown that overexpressed ADD3 and downregulated miR-145 were detected in BA liver tissues. MiR-145-5p was confirmed to target ADD3 by luciferase reporter assay. The downregulation of miR-145 may contribute to liver fibrosis in BA by upregulating the expression of ADD3.

Biliary atresia (BA) is a destructive bile duct disease occurring in newborn children within a few weeks after birth. In this study, the effect of miR-29c and miR-129-5p on epithelial-mesenchymal transition (EMT) in experimental BA was explored by constructing BA mouse models via Rhesus rotavirus vaccine infection. miR-29c and miR-129-5p expression was analyzed by real-time quantitative polymerase chain reaction. EMT was established by induction with transforming growth factor (TGF)-β1. miR-29c and miR-129-5p were overexpressed and inhibited, respectively, by Lipofectamine transfection. EMT-related protein (formin-like 2, FMNL2; E-cadherin; vimentin; and cytokeratin-19, CK-19) expression was analyzed by western blot and immunofluorescent assay. The results indicated that miR-29c and miR-129-5p were downregulated and upregulated in BA mice. TGF-β1 induction caused a time-dependent decrease and increase in miR-29c and miR-129-5p, respectively. Additionally, TGF-β1 induced an increase in FMNL2 and vimentin expression and a decrease in E-cadherin and CK-19 expression (P0.05). Overexpression or suppression of miRNA-29c or miR-129-5p, respectively, induced the inhibition of FMNL2 and vimentin, and promotion of E-cadherin and CK-19 expression, in the test groups compared to the non-intervention group (P0.05). However, the FMNL2, vimentin, E-cadherin, and CK- 19 expression did not differ between the control and non-intervention groups (P0.05). Thus, miR-29c upregulation or miR-129-5p downregulation effectively prevented EMT in BA by regulating the expression of EMT pathway-related proteins. Therefore, miR-29c and miR-129-5p could be utilized as therapeutic targets for BA in the future.