Generation of a liver specific and inducible microRNA (miR)-21 transgenic zebrafish. (A) Schematic diagram of the DNA construct used to generate LmiR21 [Tg(fabp10a-Tet^on-2A-ZsGreen:mCherry-miR-21)] transgenic zebrafish. Fabp10a promoter drives expression of ZsGreen and tetracycline-inducible transcription factor (Tet-on/off), which controls mCherry and miR-21 expression. (B) Relative quantification of miR-21 expression using (RT-qPCR) analysis. LmiR21#1-5+doxycycline (Dox) represent five independent transgenic lines. Control: wild-type (WT)-Dox and LmiR21#1-5-Dox zebrafish. (C) Liver-specific inducible miR-21 expression in the LmiR21#1 7 days post fertilization (dpf). Transgenic larvae were treated with 25 μg/mL Dox from 48 or 72 h post fertilization (hpf) to 7 dpf. Scale bar: 100 μm. (D) Left: images of zebrafish liver; right: 2D measurements of liver area using ImageJ. Scale bar: 50 μm. (E) Representative photomicrographs of IHC detection of cell proliferation marker-PCNA in zebrafish liver (Left). Scale bar: 50 μm; Statistical analysis of the percentage of PCNA-positive field. Statistically significant differences from −Dox group were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panel D and E.

Generation of a liver specific and inducible microRNA (miR)-21 transgenic zebrafish. (A) Schematic diagram of the DNA construct used to generate LmiR21 [Tg(fabp10a-Tet^on-2A-ZsGreen:mCherry-miR-21)] transgenic zebrafish. Fabp10a promoter drives expression of ZsGreen and tetracycline-inducible transcription factor (Tet-on/off), which controls mCherry and miR-21 expression. (B) Relative quantification of miR-21 expression using (RT-qPCR) analysis. LmiR21#1-5+doxycycline (Dox) represent five independent transgenic lines. Control: wild-type (WT)-Dox and LmiR21#1-5-Dox zebrafish. (C) Liver-specific inducible miR-21 expression in the LmiR21#1 7 days post fertilization (dpf). Transgenic larvae were treated with 25 μg/mL Dox from 48 or 72 h post fertilization (hpf) to 7 dpf. Scale bar: 100 μm. (D) Left: images of zebrafish liver; right: 2D measurements of liver area using ImageJ. Scale bar: 50 μm. (E) Representative photomicrographs of IHC detection of cell proliferation marker-PCNA in zebrafish liver (Left). Scale bar: 50 μm; Statistical analysis of the percentage of PCNA-positive field. Statistically significant differences from −Dox group were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panel D and E.

Characterization of hepatic steatosis in LmiR21 larvae and adult. (A) Whole-mount oil-red O (ORO) staining of WT ± Dox and LmiR21 ± Dox larvae at 21 dpf. Lipid contents in livers and swim bladders were stained by ORO (up). Scale bar: 2 mm. Percentages of WT ± Dox and LmiR21 ± Dox larvae with weak, moderate and strong levels of hepatic steatosis at 21 dpf (down). Scale bar: 200 μm. (B) Representative ORO images of liver in 21 dpf larvae (left). Scale bar: 500 μm. Hematoxylin-eosin (H&E) staining of livers at 21 dpf LmiR21 + Dox larvae show abnormally shaped hepatocytes with lipid vacuoles (arrows) compared with that in the controls at the same stage. Scale bar: 100 μm. (right). (C) Gross liver phenotypes at 4 months post fertilization (mpf) LmiR21 ± Dox. Normal hepatic morphology is clearly composed of lobes in LmiR21 − Dox adult (panels 1,2). Liver hyperplasia with yellow and greasy phenotypes in LmiR21 + Dox adult liver (panels 3,4). Scale bar: 5 mm. (D) Triglyceride (TG) and total cholesterol (TC) in the livers is greater in LmiR21 + Dox than in the controls (n = 3). Statistically significant differences from LmiR21 − Dox were denoted by ** (p < 0.01). (E) H&E staining of liver tissue from WT ± Dox and LmiR21 ± Dox at 4 mpf depicting morphological changes resulting from accumulation of microvesicular steatosis. Scale bar: 50 μm. (left). ORO staining of liver cryosections from WT ± Dox and LmiR21 ± Dox adults at 4 mpf. Scale bar: 50 μm. An abundance of lipid accumulation is observed in LmiR21 + Dox compared to that in controls. (right). (F) Representative liver sections showing ballooned hepatocytes (large arrow) and lipid vacuoles (star) with Masson’s trichrome (left), and Picrosirius red (right). Scale bar: 50 μm.

Characterization of hepatic steatosis in LmiR21 larvae and adult. (A) Whole-mount oil-red O (ORO) staining of WT ± Dox and LmiR21 ± Dox larvae at 21 dpf. Lipid contents in livers and swim bladders were stained by ORO (up). Scale bar: 2 mm. Percentages of WT ± Dox and LmiR21 ± Dox larvae with weak, moderate and strong levels of hepatic steatosis at 21 dpf (down). Scale bar: 200 μm. (B) Representative ORO images of liver in 21 dpf larvae (left). Scale bar: 500 μm. Hematoxylin-eosin (H&E) staining of livers at 21 dpf LmiR21 + Dox larvae show abnormally shaped hepatocytes with lipid vacuoles (arrows) compared with that in the controls at the same stage. Scale bar: 100 μm. (right). (C) Gross liver phenotypes at 4 months post fertilization (mpf) LmiR21 ± Dox. Normal hepatic morphology is clearly composed of lobes in LmiR21 − Dox adult (panels 1,2). Liver hyperplasia with yellow and greasy phenotypes in LmiR21 + Dox adult liver (panels 3,4). Scale bar: 5 mm. (D) Triglyceride (TG) and total cholesterol (TC) in the livers is greater in LmiR21 + Dox than in the controls (n = 3). Statistically significant differences from LmiR21 − Dox were denoted by ** (p < 0.01). (E) H&E staining of liver tissue from WT ± Dox and LmiR21 ± Dox at 4 mpf depicting morphological changes resulting from accumulation of microvesicular steatosis. Scale bar: 50 μm. (left). ORO staining of liver cryosections from WT ± Dox and LmiR21 ± Dox adults at 4 mpf. Scale bar: 50 μm. An abundance of lipid accumulation is observed in LmiR21 + Dox compared to that in controls. (right). (F) Representative liver sections showing ballooned hepatocytes (large arrow) and lipid vacuoles (star) with Masson’s trichrome (left), and Picrosirius red (right). Scale bar: 50 μm.

Characterization of hepatic steatosis in LmiR21 larvae and adult. (A) Whole-mount oil-red O (ORO) staining of WT ± Dox and LmiR21 ± Dox larvae at 21 dpf. Lipid contents in livers and swim bladders were stained by ORO (up). Scale bar: 2 mm. Percentages of WT ± Dox and LmiR21 ± Dox larvae with weak, moderate and strong levels of hepatic steatosis at 21 dpf (down). Scale bar: 200 μm. (B) Representative ORO images of liver in 21 dpf larvae (left). Scale bar: 500 μm. Hematoxylin-eosin (H&E) staining of livers at 21 dpf LmiR21 + Dox larvae show abnormally shaped hepatocytes with lipid vacuoles (arrows) compared with that in the controls at the same stage. Scale bar: 100 μm. (right). (C) Gross liver phenotypes at 4 months post fertilization (mpf) LmiR21 ± Dox. Normal hepatic morphology is clearly composed of lobes in LmiR21 − Dox adult (panels 1,2). Liver hyperplasia with yellow and greasy phenotypes in LmiR21 + Dox adult liver (panels 3,4). Scale bar: 5 mm. (D) Triglyceride (TG) and total cholesterol (TC) in the livers is greater in LmiR21 + Dox than in the controls (n = 3). Statistically significant differences from LmiR21 − Dox were denoted by ** (p < 0.01). (E) H&E staining of liver tissue from WT ± Dox and LmiR21 ± Dox at 4 mpf depicting morphological changes resulting from accumulation of microvesicular steatosis. Scale bar: 50 μm. (left). ORO staining of liver cryosections from WT ± Dox and LmiR21 ± Dox adults at 4 mpf. Scale bar: 50 μm. An abundance of lipid accumulation is observed in LmiR21 + Dox compared to that in controls. (right). (F) Representative liver sections showing ballooned hepatocytes (large arrow) and lipid vacuoles (star) with Masson’s trichrome (left), and Picrosirius red (right). Scale bar: 50 μm.

MiR-21 targets ptenb and pparaa levels in hepatocytes, which leads to nonalcoholic fatty liver disease (NAFLD) progression. (A) Schematic alignment between the mature miR-21 seed sequence (red) and (1) ptenb, (2) pparaa mRNA target sequence (blue) at the 3′-UTR. Perfect matches are indicated by a line, and G:U pairs by a colon. (left). Whole mount in situ hybridization (ISH) assays demonstrate that miR-21 significantly represses in vivo expression of (1) ptenb and (2) pparaa mRNA in AmiR + Dox, compared to that in AmiR-Dox (right). Scale bar:1 mm. (B) MiR-21 expression in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. mRNA expression levels of ptenb and pparaa mRNA liver of WT ± Dox and LmiR21 ± Dox at 4 mpf. (C) Western blot images (left) and quantitative data (right) of Ptenb, Pparaa and p-Akt in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. (D) Increased expression of lipogenic genes in response to pten suppression was greater in LmiR21 + Dox than controls (n = 3). Decreased expression of genes involved in fatty acid β-oxidation in response to pparaa suppression was greater in LmiR21 + Dox than controls (n = 3). (E) Levels of hepatic oxidative stress, MDA, and H₂O₂ in hepatic mitochondria is also greater in LmiR21 + Dox than in the controls (n = 3). (F) Bilateral-factorial effects of miR-21 in NAFLD acceleration. Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels B–E.

MiR-21 targets ptenb and pparaa levels in hepatocytes, which leads to nonalcoholic fatty liver disease (NAFLD) progression. (A) Schematic alignment between the mature miR-21 seed sequence (red) and (1) ptenb, (2) pparaa mRNA target sequence (blue) at the 3′-UTR. Perfect matches are indicated by a line, and G:U pairs by a colon. (left). Whole mount in situ hybridization (ISH) assays demonstrate that miR-21 significantly represses in vivo expression of (1) ptenb and (2) pparaa mRNA in AmiR + Dox, compared to that in AmiR-Dox (right). Scale bar:1 mm. (B) MiR-21 expression in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. mRNA expression levels of ptenb and pparaa mRNA liver of WT ± Dox and LmiR21 ± Dox at 4 mpf. (C) Western blot images (left) and quantitative data (right) of Ptenb, Pparaa and p-Akt in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. (D) Increased expression of lipogenic genes in response to pten suppression was greater in LmiR21 + Dox than controls (n = 3). Decreased expression of genes involved in fatty acid β-oxidation in response to pparaa suppression was greater in LmiR21 + Dox than controls (n = 3). (E) Levels of hepatic oxidative stress, MDA, and H₂O₂ in hepatic mitochondria is also greater in LmiR21 + Dox than in the controls (n = 3). (F) Bilateral-factorial effects of miR-21 in NAFLD acceleration. Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels B–E.

MiR-21 targets ptenb and pparaa levels in hepatocytes, which leads to nonalcoholic fatty liver disease (NAFLD) progression. (A) Schematic alignment between the mature miR-21 seed sequence (red) and (1) ptenb, (2) pparaa mRNA target sequence (blue) at the 3′-UTR. Perfect matches are indicated by a line, and G:U pairs by a colon. (left). Whole mount in situ hybridization (ISH) assays demonstrate that miR-21 significantly represses in vivo expression of (1) ptenb and (2) pparaa mRNA in AmiR + Dox, compared to that in AmiR-Dox (right). Scale bar:1 mm. (B) MiR-21 expression in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. mRNA expression levels of ptenb and pparaa mRNA liver of WT ± Dox and LmiR21 ± Dox at 4 mpf. (C) Western blot images (left) and quantitative data (right) of Ptenb, Pparaa and p-Akt in the livers of WT ± Dox and LmiR21 ± Dox at 4 mpf. (D) Increased expression of lipogenic genes in response to pten suppression was greater in LmiR21 + Dox than controls (n = 3). Decreased expression of genes involved in fatty acid β-oxidation in response to pparaa suppression was greater in LmiR21 + Dox than controls (n = 3). (E) Levels of hepatic oxidative stress, MDA, and H₂O₂ in hepatic mitochondria is also greater in LmiR21 + Dox than in the controls (n = 3). (F) Bilateral-factorial effects of miR-21 in NAFLD acceleration. Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels B–E.

Development of nonalcoholic steatohepatitis (NASH) phenotypes in LmiR-21 zebrafish at 6 mpf. (A) Gross liver images of (a) LmiR21 − Dox male, (b) LmiR21 + Dox male, (c) LmiR21 − Dox female, (d) LmiR21 + Dox female. Scale bar: 3 mm. (B) Representative images of Masson’s trichrome staining of steatohepatitis in livers of (1) LmiR21 − Dox (insets a-d) and (2) LmiR21 + Dox; Insets: (a) hepatocyte ballooning, (b) Mallory-Denk bodies (MDBs) are indicated by arrows, and (c,d) lobular inflammation is indicated by arrows. Scale bar: 50 μm. (C) Representative images of Masson’s trichrome staining of fibrotic liver tissue from LmiR21 + Dox; Insets: (a,b) MDBs concomitant with fibrosis (red arrows) and scar tissue associated with liver fibrosis (yellow arrows) and (c,d) lobular inflammation concomitant with blue collagen (blue arrows). Scale bar: 50 μm. (D) Induction of proinflammatory gene expressions in response to inflammation was significantly greater in LmiR21 + Dox than in controls (n = 3). Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01).

Development of nonalcoholic steatohepatitis (NASH) phenotypes in LmiR-21 zebrafish at 6 mpf. (A) Gross liver images of (a) LmiR21 − Dox male, (b) LmiR21 + Dox male, (c) LmiR21 − Dox female, (d) LmiR21 + Dox female. Scale bar: 3 mm. (B) Representative images of Masson’s trichrome staining of steatohepatitis in livers of (1) LmiR21 − Dox (insets a-d) and (2) LmiR21 + Dox; Insets: (a) hepatocyte ballooning, (b) Mallory-Denk bodies (MDBs) are indicated by arrows, and (c,d) lobular inflammation is indicated by arrows. Scale bar: 50 μm. (C) Representative images of Masson’s trichrome staining of fibrotic liver tissue from LmiR21 + Dox; Insets: (a,b) MDBs concomitant with fibrosis (red arrows) and scar tissue associated with liver fibrosis (yellow arrows) and (c,d) lobular inflammation concomitant with blue collagen (blue arrows). Scale bar: 50 μm. (D) Induction of proinflammatory gene expressions in response to inflammation was significantly greater in LmiR21 + Dox than in controls (n = 3). Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01).

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.

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.

Increased susceptibility of LmiR21 zebrafish to diethylnitrosamine (DEN)-induced liver fibrosis and carcinogenesis. (A) Experimental design of three independent DEN treatments and doxycycline (Dox) dosing. (B) Representative images of livers with microscopic tumors identified following hematoxylin & eosin staining (indicated with arrows) one, three, and nine months after DEN treatment. Scale bar: 100 μm. (C) Representative images of livers with microscopic injuries identified by Masson’s trichome staining at one, three, and nine months after DEN treatment. Scale bar: 50 μm. (D) Representative images of livers with microscopic injuries identified by Sirius red staining at one, three, and nine months after DEN treatment. Scale bar: 50 μm.

Increased susceptibility of LmiR21 zebrafish to diethylnitrosamine (DEN)-induced liver fibrosis and carcinogenesis. (A) Experimental design of three independent DEN treatments and doxycycline (Dox) dosing. (B) Representative images of livers with microscopic tumors identified following hematoxylin & eosin staining (indicated with arrows) one, three, and nine months after DEN treatment. Scale bar: 100 μm. (C) Representative images of livers with microscopic injuries identified by Masson’s trichome staining at one, three, and nine months after DEN treatment. Scale bar: 50 μm. (D) Representative images of livers with microscopic injuries identified by Sirius red staining at one, three, and nine months after DEN treatment. Scale bar: 50 μm.

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).

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).

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).

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).

MiR-21 expression shows a direct correlation with activation of PI3K, TGF-β and STAT3 (PTS) signaling network proteins in LmiR21 zebrafish and human nonviral hepatocellular carcinomas (HCC). (A) Expression patterns of Ptenb, p-Akt, Smad7, p-Smad3 and p-Stat3 in 10 mpf LmiR21 ± Dox liver samples after immunohistochemical (IHC) staining. Scale bar: 50 μm. Positive signal areas per field were quantified. (B) Western blot images (left) and quantitative data (right) of PTS signaling regulatory protein in 10 mpf LmiR21 ± Dox liver. (C) Human nonviral HCC samples (n = 20) were subjected to IHC of PTEN, p-AKT, SMAD7, p-SMAD3 and p-STAT3 expression. Scale bar: 50 μm. (D) miR-21 expression profiles in the surrounding normal and liver cancer tissues from nonviral HCC patients (n = 20) were analyzed by RT-qPCR. Positive correlation of miR-21 with PTS signaling regulatory genes in 60% of the nonviral HCC samples were observed. (E) Proposed mechanism by which miR-21 links the NAHCC development. MiR-21 mediates a newly discovered PI3K/Akt, TGF-β/Smad3 and Stat3 (PTS) signaling loop and feed forward loop involving Smad3/Stat3 dependent on miR-21 expression. (Abbreviations: →: activated, ─●: inhibited) Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels A, B and D.

MiR-21 expression shows a direct correlation with activation of PI3K, TGF-β and STAT3 (PTS) signaling network proteins in LmiR21 zebrafish and human nonviral hepatocellular carcinomas (HCC). (A) Expression patterns of Ptenb, p-Akt, Smad7, p-Smad3 and p-Stat3 in 10 mpf LmiR21 ± Dox liver samples after immunohistochemical (IHC) staining. Scale bar: 50 μm. Positive signal areas per field were quantified. (B) Western blot images (left) and quantitative data (right) of PTS signaling regulatory protein in 10 mpf LmiR21 ± Dox liver. (C) Human nonviral HCC samples (n = 20) were subjected to IHC of PTEN, p-AKT, SMAD7, p-SMAD3 and p-STAT3 expression. Scale bar: 50 μm. (D) miR-21 expression profiles in the surrounding normal and liver cancer tissues from nonviral HCC patients (n = 20) were analyzed by RT-qPCR. Positive correlation of miR-21 with PTS signaling regulatory genes in 60% of the nonviral HCC samples were observed. (E) Proposed mechanism by which miR-21 links the NAHCC development. MiR-21 mediates a newly discovered PI3K/Akt, TGF-β/Smad3 and Stat3 (PTS) signaling loop and feed forward loop involving Smad3/Stat3 dependent on miR-21 expression. (Abbreviations: →: activated, ─●: inhibited) Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels A, B and D.

MiR-21 expression shows a direct correlation with activation of PI3K, TGF-β and STAT3 (PTS) signaling network proteins in LmiR21 zebrafish and human nonviral hepatocellular carcinomas (HCC). (A) Expression patterns of Ptenb, p-Akt, Smad7, p-Smad3 and p-Stat3 in 10 mpf LmiR21 ± Dox liver samples after immunohistochemical (IHC) staining. Scale bar: 50 μm. Positive signal areas per field were quantified. (B) Western blot images (left) and quantitative data (right) of PTS signaling regulatory protein in 10 mpf LmiR21 ± Dox liver. (C) Human nonviral HCC samples (n = 20) were subjected to IHC of PTEN, p-AKT, SMAD7, p-SMAD3 and p-STAT3 expression. Scale bar: 50 μm. (D) miR-21 expression profiles in the surrounding normal and liver cancer tissues from nonviral HCC patients (n = 20) were analyzed by RT-qPCR. Positive correlation of miR-21 with PTS signaling regulatory genes in 60% of the nonviral HCC samples were observed. (E) Proposed mechanism by which miR-21 links the NAHCC development. MiR-21 mediates a newly discovered PI3K/Akt, TGF-β/Smad3 and Stat3 (PTS) signaling loop and feed forward loop involving Smad3/Stat3 dependent on miR-21 expression. (Abbreviations: →: activated, ─●: inhibited) Statistically significant differences from LmiR21 − Dox were denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) for panels A, B and D.