Limits...
Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4.

Huang YH, Tiao MM, Huang LT, Chuang JH, Kuo KC, Yang YL, Wang FS - PLoS ONE (2015)

Bottom Line: In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation.In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.

ABSTRACT

Background: Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives: In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods: We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results: After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions: Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.

No MeSH data available.


Related in: MedlinePlus

Overexpression of miR-29a decreased HDAC4 expression in primary HSCs.Treatment with a miR-29a mimic and HDAC4 RNAi significantly deceased HDAC4 expression in the HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549141&req=5

pone.0136453.g006: Overexpression of miR-29a decreased HDAC4 expression in primary HSCs.Treatment with a miR-29a mimic and HDAC4 RNAi significantly deceased HDAC4 expression in the HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.

Mentions: To test the effects of miR-29a inhibition on the expression of HDAC4, primary HSCs stably expressing a miR-29a mimic, antisense inhibitor, HDAC4 RNAi and scramble control were used. As expected, overexpression of miR-29a significantly downregulated the expression of α-SMA- and HDAC4- in miR-29aTg mice compared than in WT mice (Fig 5A and 5B). In contrast, after treatment with a miR-29a anti-sense inhibitor, α-SMA and HDAC4 expression was significantly upregulated in the HSCs of miR-29aTg mice. Western blotting confirmed the immunofluorescence findings (Fig 6). We then tested whether HDAC4 affected histone acetylation in activated HSCs. Our group has previously demonstrated that HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), and observed an enrichment of H3K9Ac in the miR-29a proximal promoter in a diabetic nephropathy animal model [12]. Compared to controls, a miR-29a mimic or HDAC4 RNAi significantly increased the expression of H3K9Ac (p < 0.001 and p = 0.003, respectively; Fig 7). In contrast, addition of a miR-29a anti-sense inhibitor significantly decreased H3K9Ac expression (p = 0.02; Fig 7A). Recent reports have demonstrated that miR-29 acts as a downstream inhibitor and therapeutic miR for TGF-β1/Smad3- mediated renal fibrosis [26] and myogenic differentiation [27]. As shown in Fig 7B, a miR-29a mimic or HDAC4 RNAi inhibited both expression of both Smad3 and p-Smad3 in activated HSCs.


Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4.

Huang YH, Tiao MM, Huang LT, Chuang JH, Kuo KC, Yang YL, Wang FS - PLoS ONE (2015)

Overexpression of miR-29a decreased HDAC4 expression in primary HSCs.Treatment with a miR-29a mimic and HDAC4 RNAi significantly deceased HDAC4 expression in the HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549141&req=5

pone.0136453.g006: Overexpression of miR-29a decreased HDAC4 expression in primary HSCs.Treatment with a miR-29a mimic and HDAC4 RNAi significantly deceased HDAC4 expression in the HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
Mentions: To test the effects of miR-29a inhibition on the expression of HDAC4, primary HSCs stably expressing a miR-29a mimic, antisense inhibitor, HDAC4 RNAi and scramble control were used. As expected, overexpression of miR-29a significantly downregulated the expression of α-SMA- and HDAC4- in miR-29aTg mice compared than in WT mice (Fig 5A and 5B). In contrast, after treatment with a miR-29a anti-sense inhibitor, α-SMA and HDAC4 expression was significantly upregulated in the HSCs of miR-29aTg mice. Western blotting confirmed the immunofluorescence findings (Fig 6). We then tested whether HDAC4 affected histone acetylation in activated HSCs. Our group has previously demonstrated that HDAC4 interference increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), and observed an enrichment of H3K9Ac in the miR-29a proximal promoter in a diabetic nephropathy animal model [12]. Compared to controls, a miR-29a mimic or HDAC4 RNAi significantly increased the expression of H3K9Ac (p < 0.001 and p = 0.003, respectively; Fig 7). In contrast, addition of a miR-29a anti-sense inhibitor significantly decreased H3K9Ac expression (p = 0.02; Fig 7A). Recent reports have demonstrated that miR-29 acts as a downstream inhibitor and therapeutic miR for TGF-β1/Smad3- mediated renal fibrosis [26] and myogenic differentiation [27]. As shown in Fig 7B, a miR-29a mimic or HDAC4 RNAi inhibited both expression of both Smad3 and p-Smad3 in activated HSCs.

Bottom Line: In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation.In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.

ABSTRACT

Background: Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives: In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods: We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results: After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions: Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.

No MeSH data available.


Related in: MedlinePlus