LmiR21 zebrafish develop nonalcoholic hepatocellular carcinoma (NAHCC) phenotypes at 10 months post fertilization (mpf). (A) Gross liver images from (a) LmiR21 − Dox male, (b) LmiR-21 + Dox male, (c) LmiR21 − Dox female, and (d) LmiR21 + Dox female. Scale bar: 3 mm. (B) Microscopic views of angiogenesis in livers from bright field: (a) LmiR21 − Dox (c) LmiR21 + Dox and corresponding GFP fluorescence images (b) LmiR21 − Dox (d) LmiR21 + Dox. Scale bar: 2 mm. (C) Hematoxylin & eosin stained livers from (1) LmiR21 − Dox (insets a–d) (2) LmiR21 + Dox (insets a–d) of steatohepatitis concomitant with HCC: hepatocyte ballooning, Mallory−Denk bodies (MDB), lobular inflammation and HCC (indicated by arrows). Scale bar: 50 μm. (D) Masson’s trichrome stained livers of (1) LmiR21 − Dox (insets a–d), (2) LmiR21 + Dox (insets a–d) indicative of steatohepatitis, portal and periportal fibrosis concomitant with HCC and (3) LmiR21 + Dox (insets a–d) indicative of steatohepatitis, advanced fibrosis (cirrhosis) concomitant with HCC. Scale bar: 50 μm. (E) Analysis of selected miR-21 targeted NAHCC associated genes: HMG-box transcription factor 1 (hbp1), 3-hydroxy-3-methylglutaryl-CoA reductase (hmgcr), fatty acid binding protein 7a (fabp7a), programmed cell death 4b (pdcd4b), and tissue inhibitors of metalloproteinases 3 (timp3). (1) Schematic alignment between the mature miR-21 seed sequence and the mRNA target sequence at the 3′ UTR. (2) Whole mount in situ hybridization assays demonstrate that miR-21 significantly represses expression of NAHCC associated genes in AmiR21 fish. Scale bar: 1 mm. (3) RT-qPCR analysis of the NAHCC associated genes mRNA expression in the liver of WT ± Dox and LmiR21 ± Dox at 10 mpf. Statistically significant differences from LmiR21 − Dox were denoted by ** (p < 0.01) and *** (p < 0.001).
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