Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis, as demonstrated in human liver cirrhosis, and that its downregulation influences the activation of hepatic stellate cells. In addition, both cleaved caspase-3 production and apoptosis play a role in cholestatic liver injury. However, it is unknown if miR-29 is effective in modulating the extent of injury. We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type (WT) littermates to clarify the role of miR-29 in hepatic injury and fibrogenesis, using the bile duct-ligation (BDL) mouse model. After BDL, all three members of the miR-29 family were significantly downregulated in the livers of WT mice, and miR-29b and miR-29c were significantly downregulated in the livers of the miR-29aTg mice. Liver function, as measured by alanine transaminase and aspartate transaminase activity, was significantly improved in the miR-29aTg mice than in the WT littermates, following 1 week of obstructive jaundice. In addition, overexpression of miR-29a was associated with a significant downregulation of the expression of collagen-1α1, collagen-4α1, phospho-FADD, cleaved caspase-8, cleaved caspase-3, Bax, Bcl-2, PARP, and nuclear factor-κB, as well as an upregulation of phospho-AKT expression. In addition, there were significantly fewer TUNEL-positive liver cells in the miR-29aTg group than in the WT littermates after BDL. Our results indicate that miR-29a decreases cholestatic liver injury and fibrosis after BDL, at least partially, by modulating the extrinsic rather than intrinsic pathway of apoptosis.
Fig. S1Quantitative measurement of collagen-1α1 expression by immunofluorescence staining in liver tissue of miR-29a transgenic mice and WT littermates underwent sham operation and bile duct ligation (BDL). Data are expressed as mean ± standard error in 4 samples from miR-29a transgenic mice and 4 samples from WT littermates (TIFF 76 kb)
Fig. S2Immunoreactive caspase 8 staining between miR-29aTg mice and wild type littermates. There was significantly higher caspase-8 immunoreactivity in the liver tissue of the sham-operated and BDL groups than in the control group, which was expressed constantly in the cytoplasm of hepatocytes (arrowhead). Moreover, overexpression of miR-29a decreased the expression of caspase-8 and caspase-3 after BDL (TIFF 1900 kb)
Fig. S3Comparison of the X-linked inhibitor of the apoptotic protein (XIAP) expression by western blot in liver tissue of miR-29a transgenic mice and WT littermates that underwent sham operation and bile duct ligation (BDL). There were significantly downregulated XIAP protein expression in both miR-29aTg mice and WT littermates after BDL. However, overexpression did not change of XIAP protein expression after BDL. Data are expressed as mean ± standard error in 8 samples from miR-29a transgenic mice and 6 samples from WT littermates (TIFF 595 kb)