MiR-21 Simultaneously Regulates ERK1 Signaling in HSC Activation and Hepatocyte EMT in Hepatic Fibrosis

<div><p>Background</p><p>MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on <i>SPRY2</i> and <i>HNF4α</i> in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes.</p><p>Methods</p><p>Quantitative RT-PCR was used to determine miR-21 and the expression of <i>SPRY2, HNF4α</i> and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes <i>SPRY2</i> and <i>HNF4α</i>. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and <i>SPRY2</i> or <i>HNF4α.</i></p><p>Results</p><p>The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. <i>SPRY2</i> and <i>HNF4α</i> mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed <i>SPRY2</i> and <i>HNF4α</i> expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting <i>SPRY2</i> or <i>HNF4α</i>. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFβ1-treated hepatocytes.</p><p>Conclusions</p><p>MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating <i>SPRY2</i> and <i>HNF4α</i> expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.</p></div

Clinical features of the patients providing cirrhotic liver tissues.

<p>Clinical features of the patients providing cirrhotic liver tissues.</p

MiR-21 and <i>HNF4α</i> mutually modulate the expression of each other in regulating EMT of primary hepatocytes.

<p>Primary rat hepatocytes were treated with miR-21 mimics for 48 h. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, EMT associated genes <i>E-cadherin</i>, <i>vimentin</i> and <i>slug</i> (A), <i>MMP-2</i> and <i>MMP-9</i>, fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i> and liver specific genes <i>ALB</i>, <i>CYP1a2</i> and <i>AFP</i> (B) were examined by quantitative RT-PCR. <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, ALB, <i>MMP-2</i> and <i>TIMP1</i> were examined by Western blotting assays (C). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. Primary hepatocytes were treated with TGFβ1 for 48 h followed by AdHNF4α infection 48 h later (D). Primary hepatocytes were transfected with miR-21 mimics followed by AdHNF4α (E) or AdshHNF4α (F) infection 48 h later. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i> were then examined by quantitative RT-PCR. Each value in (A), (B), (D), (E) and (F) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments and miR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p

MiR-21 inhibitors target ERK1 signaling and <i>SPRY2</i> in HSC activation and block EMT of TGFβ1-treated hepatocytes by targeting <i>HNF4α</i>.

<p>Primary HSCs were treated with TGFβ1 for 5 d followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i> (A), <i>α-SMA</i>, <i>TIMP1</i>, <i>MMP-2</i> and <i>MMP-13</i> (B) were examined by quantitative RT-PCR. Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i> (C). HSC-T6 cells were transfected with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>SRPY2</i>, <i>ERK1</i> and <i>RSK2</i>, <i>MMP-9</i>, <i>MMP-13</i>, fibrogenesis-associated genes <i>TGFβ1</i>, α<i>-SMA</i>, <i>Col I</i>, <i>Col III</i> and <i>TIMP1</i> were examined by quantitative RT-PCR (D, E). Immunoblotting assays were done to detect the expression of <i>SPRY2</i>, <i>phospho-ERK1</i>, <i>phospho-ERK2</i> and <i>RSK2</i>, <i>Col I</i> and <i>TIMP1</i> (F). HSC-T6 cells were transfected with miR-21 inhibitors and transwell cell migration assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a> (G, H). Hepatocytes were treated with TGFβ1 for 48 h followed by transfection with miR-21 inhibitors. MiR-21 and the mRNA transcript levels of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP-2</i>, <i>MMP-9</i> and α<i>-SMA</i>, liver specific genes <i>CYP1a2</i> and <i>AFP</i> were examined by quantitative RT-PCR (I, J). Immunoblotting assays were done to detect the expression of <i>HNF4α</i>, <i>E-cadherin</i> and <i>vimentin</i>, <i>MMP2</i>, <i>α-SMA</i> and <i>TIMP1</i> (K). GADPH was used as a loading control. Blots representative of at least three independent experiments are shown. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. Each value in (A), (B), (D), (E), (H), (I) and (J) represents the mean with the SD (error bars) for triplicate samples of at least three independent experiments. *<i>P</i><0.05 or **<i>P</i><0.01 vs. controls.</p

Liver fibrosis is associated with broad changes in the expression of miR-21 and its targeted genes, ERK1 signaling and EMT-associated genes.

<p>Serum miR-21 contents in cirrhotic patients (n = 20) and normal subjects (n = 20) (A) and in rats with dimethylnitrosamine-induced liver cirrhosis (B). MiR-21 expression was normalized against miR-238 in serum. **<i>P</i><0.01 vs. normal controls. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α</i>, <i>ERK1</i> and its downstream target <i>RSK2</i>, and epithelial-mesenchymal transition (EMT)-associated genes <i>E-cadherin</i> and <i>vimentin</i> in the liver tissues of cirrhotic patients (n = 7) (C) and rats (n = 10) (D). *<i>P</i><0.05 and **<i>P</i><0.01 vs. normal subjects or rats. Primary HSCs were treated with TGFβ1 for 7 days (E) and primary hepatocytes for 48 h (F) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108005#s2" target="_blank">methods</a>. Quantitative RT-PCR examination of the expression of miR-21 and its targeted genes <i>SPRY2</i> and <i>HNF4α, ERK1</i> and <i>RSK2</i>, <i>E-cadherin</i>, <i>vimentin</i> and <i>MMP-9</i>, liver fibrogenesis-associated genes α<i>-SMA</i> and <i>TIMP1</i>, and liver specific genes <i>ALB</i> and <i>CYP1a2</i>. MiR-21 expression was normalized against <i>Rnu6-2</i> and mRNA expression was normalized against <i>β-actin</i>. *<i>P</i><0.05 and **<i>P</i><0.01 vs. non-stimulated HSCs or hepatocytes. Each value represents the mean with the SD (error bars) for triplicate samples.</p