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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.

No MeSH data available.


Related in: MedlinePlus

miR-706 has a minor effect on oxidative stress-induced apoptosis in hepatocytes.H2O2 (300 uM) induced decreased cell viability measured with CCK8 in hepatic L02 cells, *P < 0.05. n = 4. (B) Introduction of miR-706 has little effect on cell viability in hepatic L02 cells. L02 cells were transfected with negative control (NC) or miR-706 (at a concentration of 12.5 nM, 25 nM, 50 nM and 100 nM, respectively) duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 6 h before measurement with CCK8, n = 4. (C) Hepatic L02 cells were transfected with negative control (NC) or miR-706 duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 1 h and 24 h, and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad were measured by real-time PCR. GAPDH was used as an internal control. * P < 0.05. (D) mRNA expression of Bax, Bak1, Bbc3, and Bad were detected by real-time PCR and (E) cleaved caspase-3 expression was detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. GAPDH was used as an internal control. *P < 0.05.
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f6: miR-706 has a minor effect on oxidative stress-induced apoptosis in hepatocytes.H2O2 (300 uM) induced decreased cell viability measured with CCK8 in hepatic L02 cells, *P < 0.05. n = 4. (B) Introduction of miR-706 has little effect on cell viability in hepatic L02 cells. L02 cells were transfected with negative control (NC) or miR-706 (at a concentration of 12.5 nM, 25 nM, 50 nM and 100 nM, respectively) duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 6 h before measurement with CCK8, n = 4. (C) Hepatic L02 cells were transfected with negative control (NC) or miR-706 duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 1 h and 24 h, and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad were measured by real-time PCR. GAPDH was used as an internal control. * P < 0.05. (D) mRNA expression of Bax, Bak1, Bbc3, and Bad were detected by real-time PCR and (E) cleaved caspase-3 expression was detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. GAPDH was used as an internal control. *P < 0.05.

Mentions: Previous studies have described oxidative stress-induced apoptosis of L02 cells36. In the current study, decreased cell viability was detected by H2O2 treatment from 6 h to 48 h (Fig. 6A), however, a certain proportion of cells still survived. These results suggest that surviving cells might undergo EMT. Then we investigated the effects of miR-706 on hepatocyte apoptosis. We found that miR-706 had little effect on H2O2-induced decreased hepatocyte cell viability and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad (Fig. 6B,C). In our in vivo study, we found that CCl4 treatment induced increased expression of the pro-apoptotic genes Bax, Bak1, Bbc3, and Bad. Among them, Bax and Bad can be inhibited by adding miR-706 agomir. However, miR-706 agomir did not prevent the increase in expression of Bak1 and Bbc3 induced by CCl4 treatment (Fig. 6D). Furthermore, CCl4 treatment induced increased cleaved Caspase-3 expression, which was not alleviated by miR-706 agomir injection (Fig. 6E). These results indicate that miR-706 may inhibit some pro-apoptotic genes during liver fibrosis, but have no direct effect on overall hepatocyte apoptosis. Further mechanisms need to be investigated in future studies.


miR-706 inhibits the oxidative stress-induced activation of PKC α /TAOK1 in liver fibrogenesis
miR-706 has a minor effect on oxidative stress-induced apoptosis in hepatocytes.H2O2 (300 uM) induced decreased cell viability measured with CCK8 in hepatic L02 cells, *P < 0.05. n = 4. (B) Introduction of miR-706 has little effect on cell viability in hepatic L02 cells. L02 cells were transfected with negative control (NC) or miR-706 (at a concentration of 12.5 nM, 25 nM, 50 nM and 100 nM, respectively) duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 6 h before measurement with CCK8, n = 4. (C) Hepatic L02 cells were transfected with negative control (NC) or miR-706 duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 1 h and 24 h, and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad were measured by real-time PCR. GAPDH was used as an internal control. * P < 0.05. (D) mRNA expression of Bax, Bak1, Bbc3, and Bad were detected by real-time PCR and (E) cleaved caspase-3 expression was detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. GAPDH was used as an internal control. *P < 0.05.
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f6: miR-706 has a minor effect on oxidative stress-induced apoptosis in hepatocytes.H2O2 (300 uM) induced decreased cell viability measured with CCK8 in hepatic L02 cells, *P < 0.05. n = 4. (B) Introduction of miR-706 has little effect on cell viability in hepatic L02 cells. L02 cells were transfected with negative control (NC) or miR-706 (at a concentration of 12.5 nM, 25 nM, 50 nM and 100 nM, respectively) duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 6 h before measurement with CCK8, n = 4. (C) Hepatic L02 cells were transfected with negative control (NC) or miR-706 duplex for 8 h, and then stimulated with 300 μM H2O2 or remained untreated for 1 h and 24 h, and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad were measured by real-time PCR. GAPDH was used as an internal control. * P < 0.05. (D) mRNA expression of Bax, Bak1, Bbc3, and Bad were detected by real-time PCR and (E) cleaved caspase-3 expression was detected by Western blot in 6-week CCl4-treated livers injected with miR-706 agomir. GAPDH was used as an internal control. *P < 0.05.
Mentions: Previous studies have described oxidative stress-induced apoptosis of L02 cells36. In the current study, decreased cell viability was detected by H2O2 treatment from 6 h to 48 h (Fig. 6A), however, a certain proportion of cells still survived. These results suggest that surviving cells might undergo EMT. Then we investigated the effects of miR-706 on hepatocyte apoptosis. We found that miR-706 had little effect on H2O2-induced decreased hepatocyte cell viability and pro-apoptotic genes Bax, Bak1, Bbc3, and Bad (Fig. 6B,C). In our in vivo study, we found that CCl4 treatment induced increased expression of the pro-apoptotic genes Bax, Bak1, Bbc3, and Bad. Among them, Bax and Bad can be inhibited by adding miR-706 agomir. However, miR-706 agomir did not prevent the increase in expression of Bak1 and Bbc3 induced by CCl4 treatment (Fig. 6D). Furthermore, CCl4 treatment induced increased cleaved Caspase-3 expression, which was not alleviated by miR-706 agomir injection (Fig. 6E). These results indicate that miR-706 may inhibit some pro-apoptotic genes during liver fibrosis, but have no direct effect on overall hepatocyte apoptosis. Further mechanisms need to be investigated in future studies.

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