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Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice

View Article: PubMed Central - PubMed

ABSTRACT

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to play a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR21−/− mice underwent sham or bile duct ligation (BDL) for 1 wk, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and Smad-7 expression. In vitro, immortalized murine biliary cell lines (IMCL) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. Also, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers TGF-β1 and α-SMA. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared to control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.

No MeSH data available.


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Evaluation of HSC activation and fibrotic reaction. As shown by immunofluorescence co-stain for SYP-9 (green) and CK-19 (red) there is increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− mice when compared to BDL WT, respectively (A). Immunofluorescent co-stain for α-SMA (green) and CK-19 (red) show increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− when compared to BDL WT (B). In HSCs that are isolated from BDL WT there is a significant increase in Collagen-1a expression when compared to HSCs isolated from Sham WT; however, Collagen-1a expression is significantly decreased in HSCs isolated from BDL miR-21−/− when compared to HSCs isolated from BDL WT (C). Data are expressed as means ± SEM. n=6 reactions in total RNA from 6 animals per group for qPCR. *p<0.05 vs. Sham WT; #p<0.05 vs. BDL WT. Representative images are shown for SYP-9 and CK-19 immunofluorescence. Original magnification, 20X.
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Figure 5: Evaluation of HSC activation and fibrotic reaction. As shown by immunofluorescence co-stain for SYP-9 (green) and CK-19 (red) there is increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− mice when compared to BDL WT, respectively (A). Immunofluorescent co-stain for α-SMA (green) and CK-19 (red) show increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− when compared to BDL WT (B). In HSCs that are isolated from BDL WT there is a significant increase in Collagen-1a expression when compared to HSCs isolated from Sham WT; however, Collagen-1a expression is significantly decreased in HSCs isolated from BDL miR-21−/− when compared to HSCs isolated from BDL WT (C). Data are expressed as means ± SEM. n=6 reactions in total RNA from 6 animals per group for qPCR. *p<0.05 vs. Sham WT; #p<0.05 vs. BDL WT. Representative images are shown for SYP-9 and CK-19 immunofluorescence. Original magnification, 20X.

Mentions: Since the activation of HSCs is known to be a key player in the development of fibrosis 36, we analyzed HSC activation in vivo by staining for SYP-9 or α-SMA (markers of activated HSCs) and CK-19 (to visualize bile ducts) in liver sections by immunofluorescence. The expression of SYP-9 and CK-19 seemed unchanged between Sham WT and Sham miR-21−/− mice; however, the number of SYP-9 positive cells was increased in BDL WT, which was accompanied with increased IBDM. In BDL miR21−/− mice the number of SYP-9 positive cells was decreased compared to BDL WT, and this was also accompanied by decreased IBDM (Figure 5A). Staining for α-SMA and CK-19 showed the same trend as the SYP-9 staining (Figure 5B). Recently, studies have indicated that miR-21 plays a pro-fibrotic role during models of drug-induced cholestasis, and that inhibition of miR-21 can ameliorate this damage 37, 38. In HSCs that were isolated from BDL WT we see increased expression of Collagen-1a when compared to Sham WT (Figure 5C). In contrast, in HSCs that were isolated from BDL miR-21−/− these parameters are decreased when compared to BDL WT (Figure 5C). No significant differences were noted between HSCs isolated from Sham WT and Sham miR-21−/−. These data further imply that increased miR-21 levels can contribute to increased fibrosis via HSC activation and fibrotic reaction.


Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice
Evaluation of HSC activation and fibrotic reaction. As shown by immunofluorescence co-stain for SYP-9 (green) and CK-19 (red) there is increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− mice when compared to BDL WT, respectively (A). Immunofluorescent co-stain for α-SMA (green) and CK-19 (red) show increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− when compared to BDL WT (B). In HSCs that are isolated from BDL WT there is a significant increase in Collagen-1a expression when compared to HSCs isolated from Sham WT; however, Collagen-1a expression is significantly decreased in HSCs isolated from BDL miR-21−/− when compared to HSCs isolated from BDL WT (C). Data are expressed as means ± SEM. n=6 reactions in total RNA from 6 animals per group for qPCR. *p<0.05 vs. Sham WT; #p<0.05 vs. BDL WT. Representative images are shown for SYP-9 and CK-19 immunofluorescence. Original magnification, 20X.
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Figure 5: Evaluation of HSC activation and fibrotic reaction. As shown by immunofluorescence co-stain for SYP-9 (green) and CK-19 (red) there is increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− mice when compared to BDL WT, respectively (A). Immunofluorescent co-stain for α-SMA (green) and CK-19 (red) show increased HSC activation and IBDM in BDL WT compared to Sham WT, but this is decreased in BDL miR-21−/− when compared to BDL WT (B). In HSCs that are isolated from BDL WT there is a significant increase in Collagen-1a expression when compared to HSCs isolated from Sham WT; however, Collagen-1a expression is significantly decreased in HSCs isolated from BDL miR-21−/− when compared to HSCs isolated from BDL WT (C). Data are expressed as means ± SEM. n=6 reactions in total RNA from 6 animals per group for qPCR. *p<0.05 vs. Sham WT; #p<0.05 vs. BDL WT. Representative images are shown for SYP-9 and CK-19 immunofluorescence. Original magnification, 20X.
Mentions: Since the activation of HSCs is known to be a key player in the development of fibrosis 36, we analyzed HSC activation in vivo by staining for SYP-9 or α-SMA (markers of activated HSCs) and CK-19 (to visualize bile ducts) in liver sections by immunofluorescence. The expression of SYP-9 and CK-19 seemed unchanged between Sham WT and Sham miR-21−/− mice; however, the number of SYP-9 positive cells was increased in BDL WT, which was accompanied with increased IBDM. In BDL miR21−/− mice the number of SYP-9 positive cells was decreased compared to BDL WT, and this was also accompanied by decreased IBDM (Figure 5A). Staining for α-SMA and CK-19 showed the same trend as the SYP-9 staining (Figure 5B). Recently, studies have indicated that miR-21 plays a pro-fibrotic role during models of drug-induced cholestasis, and that inhibition of miR-21 can ameliorate this damage 37, 38. In HSCs that were isolated from BDL WT we see increased expression of Collagen-1a when compared to Sham WT (Figure 5C). In contrast, in HSCs that were isolated from BDL miR-21−/− these parameters are decreased when compared to BDL WT (Figure 5C). No significant differences were noted between HSCs isolated from Sham WT and Sham miR-21−/−. These data further imply that increased miR-21 levels can contribute to increased fibrosis via HSC activation and fibrotic reaction.

View Article: PubMed Central - PubMed

ABSTRACT

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to play a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR21&minus;/&minus; mice underwent sham or bile duct ligation (BDL) for 1 wk, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and Smad-7 expression. In vitro, immortalized murine biliary cell lines (IMCL) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. Also, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers TGF-&beta;1 and &alpha;-SMA. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared to control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.

No MeSH data available.


Related in: MedlinePlus