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miR-706 inhibits the oxidative stress-induced activation of PKC α /TAOK1 in liver fibrogenesis

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress induces the activation of liver fibrogenic cells (myofibroblasts), thus promoting the expression of fibrosis-related genes, leading to hepatic fibrogenesis. MicroRNAs (miRNAs) are a new class of small RNAs ~18–25 nucleotides in length involved in post-transcriptional regulation of gene expression. Wound-healing and remodeling processes in liver fibrosis have been associated with changes in hepatic miRNA expression. However, the role of miR-706 in liver fibrogenesis is currently unknown. In the present study, we show that miR-706 is abundantly expressed in hepatocytes. Moreover, oxidative stress leads to a significant downregulation of miR-706, and the further reintroduction of miR-706 inhibits oxidative stress-induced expression of fibrosis-related markers such as α-SMA. Subsequent studies revealed that miR-706 directly inhibits PKCα and TAOK1 expression via binding to the 3′-untranslated region, preventing epithelial mesenchymal transition. In vivo studies showed that intravenous injection of miR-706 agomir successfully increases hepatic miR-706 and decreases α-SMA, PKCα, and TAOK1 protein levels in livers of carbon tetrachloride (CCl4)-treated mice. In summary, this study reveals a protective role for miR-706 by blocking the oxidative stress-induced activation of PKCα/TAOK1. Our results further identify a major implication for miR-706 in preventing hepatic fibrogenesis and suggest that miR-706 may be a suitable molecular target for anti-fibrosis therapy.

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Gene transfer of miR-706 prevents CCl4-induced liver fibrosis in mice.(A) Administration of miR-706 agomir enhanced miR-706 levels in livers analyzed by RT-PCR after 1 week of the second injection of miR-706 agomir, *P < 0.05. (B) Quantification of hepatic hydroxyproline content; the data are expressed as hydroxyproline (μg)/liver wet weight (mg). *P < 0.05. (C) Decreased α-SMA, Col1a1 expression, and Sirius Red staining were detected by immunohistochemistry staining in 6-week CCl4-treated livers injected with miR-706 agomir. (D) Quantification of the Sirius red-positive area. *P < 0.05. (E) mRNA expression of α-SMA, col1a1, prkca and Taok1 were detected by real-time PCR. GAPDH was used as an internal control. *P < 0.05. (F) Decreased α-SMA and Col1 expression and inhibited PKCα and TAOK1 were detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. The levels of target genes in each sample were normalized to that of GAPDH (internal control). *P < 0.05.
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f5: Gene transfer of miR-706 prevents CCl4-induced liver fibrosis in mice.(A) Administration of miR-706 agomir enhanced miR-706 levels in livers analyzed by RT-PCR after 1 week of the second injection of miR-706 agomir, *P < 0.05. (B) Quantification of hepatic hydroxyproline content; the data are expressed as hydroxyproline (μg)/liver wet weight (mg). *P < 0.05. (C) Decreased α-SMA, Col1a1 expression, and Sirius Red staining were detected by immunohistochemistry staining in 6-week CCl4-treated livers injected with miR-706 agomir. (D) Quantification of the Sirius red-positive area. *P < 0.05. (E) mRNA expression of α-SMA, col1a1, prkca and Taok1 were detected by real-time PCR. GAPDH was used as an internal control. *P < 0.05. (F) Decreased α-SMA and Col1 expression and inhibited PKCα and TAOK1 were detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. The levels of target genes in each sample were normalized to that of GAPDH (internal control). *P < 0.05.

Mentions: We next investigated whether reintroduction of miR-706 could attenuate CCl4-induced hepatic fibrosis in vivo. After 4 weeks of CCl4 treatment, mice were injected intravenously either with control agomir or miR-706 agomir once per week for a period of 2 weeks. Injection of miR-706 agomir increased miR-706 levels in liver tissue (Fig. 5A). Quantitative analysis revealed that miR-706 significantly decreased hydroxyproline content compared to control agomir (Fig. 5B), and greatly attenuated the severity of liver fibrosis, as demonstrated by α-SMA, Col1a1, Sirius red staining (Fig. 5C,D). Moreover, Western blot analysis indicated that treatment with miR-706 agomir markedly inhibited α-SMA, Col1a1, PKCα, and TAOK1 expression in livers of CCl4-treated mice compared with those injected with control agomir (Fig. 5F). These data indicated that miR-706 may attenuate liver fibrosis progression to some extent. Furthermore, treatment with miR-706 agomir significantly reduced the mRNA levels of α-SMA, Col1a1, Prkca, and Taok1 in the livers of CCl4-treated mice (Fig. 5E). Furthermore, the injection of miR-706 antagomir promoted liver fibrosis characterized by increased α-SMA and Col1 expression, associated with high PKCα and TAOK1 expression (Supporting Fig. 5). All together, these results indicated that miR-706 may attenuate hepatic fibrogenesis in vivo via downregulation of PKCα and TAOK1 expression.


miR-706 inhibits the oxidative stress-induced activation of PKC α /TAOK1 in liver fibrogenesis
Gene transfer of miR-706 prevents CCl4-induced liver fibrosis in mice.(A) Administration of miR-706 agomir enhanced miR-706 levels in livers analyzed by RT-PCR after 1 week of the second injection of miR-706 agomir, *P < 0.05. (B) Quantification of hepatic hydroxyproline content; the data are expressed as hydroxyproline (μg)/liver wet weight (mg). *P < 0.05. (C) Decreased α-SMA, Col1a1 expression, and Sirius Red staining were detected by immunohistochemistry staining in 6-week CCl4-treated livers injected with miR-706 agomir. (D) Quantification of the Sirius red-positive area. *P < 0.05. (E) mRNA expression of α-SMA, col1a1, prkca and Taok1 were detected by real-time PCR. GAPDH was used as an internal control. *P < 0.05. (F) Decreased α-SMA and Col1 expression and inhibited PKCα and TAOK1 were detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. The levels of target genes in each sample were normalized to that of GAPDH (internal control). *P < 0.05.
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f5: Gene transfer of miR-706 prevents CCl4-induced liver fibrosis in mice.(A) Administration of miR-706 agomir enhanced miR-706 levels in livers analyzed by RT-PCR after 1 week of the second injection of miR-706 agomir, *P < 0.05. (B) Quantification of hepatic hydroxyproline content; the data are expressed as hydroxyproline (μg)/liver wet weight (mg). *P < 0.05. (C) Decreased α-SMA, Col1a1 expression, and Sirius Red staining were detected by immunohistochemistry staining in 6-week CCl4-treated livers injected with miR-706 agomir. (D) Quantification of the Sirius red-positive area. *P < 0.05. (E) mRNA expression of α-SMA, col1a1, prkca and Taok1 were detected by real-time PCR. GAPDH was used as an internal control. *P < 0.05. (F) Decreased α-SMA and Col1 expression and inhibited PKCα and TAOK1 were detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. The levels of target genes in each sample were normalized to that of GAPDH (internal control). *P < 0.05.
Mentions: We next investigated whether reintroduction of miR-706 could attenuate CCl4-induced hepatic fibrosis in vivo. After 4 weeks of CCl4 treatment, mice were injected intravenously either with control agomir or miR-706 agomir once per week for a period of 2 weeks. Injection of miR-706 agomir increased miR-706 levels in liver tissue (Fig. 5A). Quantitative analysis revealed that miR-706 significantly decreased hydroxyproline content compared to control agomir (Fig. 5B), and greatly attenuated the severity of liver fibrosis, as demonstrated by α-SMA, Col1a1, Sirius red staining (Fig. 5C,D). Moreover, Western blot analysis indicated that treatment with miR-706 agomir markedly inhibited α-SMA, Col1a1, PKCα, and TAOK1 expression in livers of CCl4-treated mice compared with those injected with control agomir (Fig. 5F). These data indicated that miR-706 may attenuate liver fibrosis progression to some extent. Furthermore, treatment with miR-706 agomir significantly reduced the mRNA levels of α-SMA, Col1a1, Prkca, and Taok1 in the livers of CCl4-treated mice (Fig. 5E). Furthermore, the injection of miR-706 antagomir promoted liver fibrosis characterized by increased α-SMA and Col1 expression, associated with high PKCα and TAOK1 expression (Supporting Fig. 5). All together, these results indicated that miR-706 may attenuate hepatic fibrogenesis in vivo via downregulation of PKCα and TAOK1 expression.

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress induces the activation of liver fibrogenic cells (myofibroblasts), thus promoting the expression of fibrosis-related genes, leading to hepatic fibrogenesis. MicroRNAs (miRNAs) are a new class of small RNAs ~18&ndash;25 nucleotides in length involved in post-transcriptional regulation of gene expression. Wound-healing and remodeling processes in liver fibrosis have been associated with changes in hepatic miRNA expression. However, the role of miR-706 in liver fibrogenesis is currently unknown. In the present study, we show that miR-706 is abundantly expressed in hepatocytes. Moreover, oxidative stress leads to a significant downregulation of miR-706, and the further reintroduction of miR-706 inhibits oxidative stress-induced expression of fibrosis-related markers such as &alpha;-SMA. Subsequent studies revealed that miR-706 directly inhibits PKC&alpha; and TAOK1 expression via binding to the 3&prime;-untranslated region, preventing epithelial mesenchymal transition. In vivo studies showed that intravenous injection of miR-706 agomir successfully increases hepatic miR-706 and decreases &alpha;-SMA, PKC&alpha;, and TAOK1 protein levels in livers of carbon tetrachloride (CCl4)-treated mice. In summary, this study reveals a protective role for miR-706 by blocking the oxidative stress-induced activation of PKC&alpha;/TAOK1. Our results further identify a major implication for miR-706 in preventing hepatic fibrogenesis and suggest that miR-706 may be a suitable molecular target for anti-fibrosis therapy.

No MeSH data available.


Related in: MedlinePlus