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Figure 5 MiR-21 targets smad7 and ptenb in hepatocytes and leads to fibrogenesis. (A) Schematic alignment between the mature miR-21 seed sequence (red) and the smad7 mRNA target sequence (blue) at the 3-UTR. Perfect matches are indicated by a line, and G:U pairs by a colon. Whole mount in situ hybridization assays show significant repression of smad7 mRNA by miR-21 in AmiR+Dox, as compared to AmiR21 − Dox. Scale bar: 1 mm. (B) RT-qPCR analysis of smad7 and ptenb mRNA expression in the liver of WT ± Dox and LmiR21 ± Dox fish at 8 mpf. (C) Representative Western blots of Smad7 and PTEN in livers of WT ± Dox and LmiR21 ± Dox at 8 mpf. (D) Representative images of Masson’s trichrome and Picrosirius red staining of liver tissue from WT±Dox and LmiR21 ± Dox at 8 mpf depicting the individual components of advanced fibrosis (as indicated): portal and periportal fibrosis, bridging fibrosis and cirrhosis. Scale bar: 50 μm. (E) Increased expression of HSC and fibrotic genes in response to Smad7 and PTEN suppression was significantly greater in LmiR21 + Dox at 8 mpf than controls (n = 3). (F) Bilateral-factorial effects of miR-21 in fibrogenesis activation. Statistically significant differences from LmiR21 − Dox are denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels B, C and E.