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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 hHSC miR-21 proliferation, apoptosis and fibrotic reaction, in vitro. hHSCs treated with miR-21 inhibitor have decreased proliferation, increased Bax, and decreased α-SMA and MMP-9 gene expression when compared to control treated (B, C, D, E). Data are expressed as means ± SEM. n = 6 reactions from 12 sets of cells for qPCR, n = 10 experiments for MTS assay. *p<0.05 versus hHSC control.
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Figure 7: Evaluation of hHSC miR-21 proliferation, apoptosis and fibrotic reaction, in vitro. hHSCs treated with miR-21 inhibitor have decreased proliferation, increased Bax, and decreased α-SMA and MMP-9 gene expression when compared to control treated (B, C, D, E). Data are expressed as means ± SEM. n = 6 reactions from 12 sets of cells for qPCR, n = 10 experiments for MTS assay. *p<0.05 versus hHSC control.

Mentions: As demonstrated above, miR-21 seems to have a role in HSC activation and proliferation following bile duct ligation in vivo (Figure 5). To further validate these findings, we took hHSCs and treated them with either miR-21 inhibitor or control. Following treatment, proliferative capacity was decreased in hHSC treated with a miR-21 inhibitor when compared to hHSC treated with control as demonstrated by MTS assay and PCNA expression (Figure 7A and 7B). Concomitantly, hHSCs treated with miR-21 inhibitor have increased Bax expression when compared to control treated cells (Figure 7C) indicating that the loss of miR-21 decreases HSC proliferation. We wanted to look further at the role of miR-21 during HSC activation since we previously noted that BDL miR-21−/− mice have decreased HSC activation when compared to BDL WT mice (Figure 5). By qPCR, we found that hHSCs treated with miR-21 inhibitor had decreased expression of α-SMA and MMP-9 when compared to control (Figure 7D). These findings suggest that the loss of miR-21 decreases the degree of HSC proliferation and fibrotic reaction, which could be an important target to halt cholestasis-induced hepatic fibrosis progression.


Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice
Evaluation of hHSC miR-21 proliferation, apoptosis and fibrotic reaction, in vitro. hHSCs treated with miR-21 inhibitor have decreased proliferation, increased Bax, and decreased α-SMA and MMP-9 gene expression when compared to control treated (B, C, D, E). Data are expressed as means ± SEM. n = 6 reactions from 12 sets of cells for qPCR, n = 10 experiments for MTS assay. *p<0.05 versus hHSC control.
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Figure 7: Evaluation of hHSC miR-21 proliferation, apoptosis and fibrotic reaction, in vitro. hHSCs treated with miR-21 inhibitor have decreased proliferation, increased Bax, and decreased α-SMA and MMP-9 gene expression when compared to control treated (B, C, D, E). Data are expressed as means ± SEM. n = 6 reactions from 12 sets of cells for qPCR, n = 10 experiments for MTS assay. *p<0.05 versus hHSC control.
Mentions: As demonstrated above, miR-21 seems to have a role in HSC activation and proliferation following bile duct ligation in vivo (Figure 5). To further validate these findings, we took hHSCs and treated them with either miR-21 inhibitor or control. Following treatment, proliferative capacity was decreased in hHSC treated with a miR-21 inhibitor when compared to hHSC treated with control as demonstrated by MTS assay and PCNA expression (Figure 7A and 7B). Concomitantly, hHSCs treated with miR-21 inhibitor have increased Bax expression when compared to control treated cells (Figure 7C) indicating that the loss of miR-21 decreases HSC proliferation. We wanted to look further at the role of miR-21 during HSC activation since we previously noted that BDL miR-21−/− mice have decreased HSC activation when compared to BDL WT mice (Figure 5). By qPCR, we found that hHSCs treated with miR-21 inhibitor had decreased expression of α-SMA and MMP-9 when compared to control (Figure 7D). These findings suggest that the loss of miR-21 decreases the degree of HSC proliferation and fibrotic reaction, which could be an important target to halt cholestasis-induced hepatic fibrosis progression.

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