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Effect of miR-34a in regulating steatosis by targeting PPARα expression in nonalcoholic fatty liver disease.

Ding J, Li M, Wan X, Jin X, Chen S, Yu C, Li Y - Sci Rep (2015)

Bottom Line: The upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 that are the direct targets of miR-34a.Activation of the central metabolic sensor AMPK was also increased.The miR-34a inhibitor suppressed lipid accumulation and improved the degree of steatosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Hangzhou First People's Hospital, No. 261 Huansha Road, Hangzhou 310006, Zhejiang Province, China.

ABSTRACT
MicroRNA-34a (miR-34a) is thought to be involved in nonalcoholic fatty liver disease (NAFLD). However, the association between altered expression of miR-34a and the pathophysiological features of NAFLD remains unclear. Here, we investigated the mechanisms by which miR-34a influences NAFLD through the PPARα-related pathway. Real-time quantitative PCR, western blotting and other assays kit were used to investigate the expression and function of miR-34a in an NAFLD model. Cultured cells transfected with miR-34a inhibitor and C57BL/6 mice injected with the miR-34a inhibitor through vein tail were conducted for the effects of miR-34a on its target. MiR-34a levels were significantly upregulated in steatosis-induced hepatocytes and in liver tissues of high-fat diet-fed mice. The upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 that are the direct targets of miR-34a. Silencing miR-34a led to an initially increased expression of PPARα, SIRT1 and PPARα's downstream genes. Activation of the central metabolic sensor AMPK was also increased. The miR-34a inhibitor suppressed lipid accumulation and improved the degree of steatosis. Taken together, our data indicated that decreased expression of miR-34a potentially contributes to altered lipid metabolism in NAFLD. Downregulation of miR-34a may be a therapeutic strategy against NAFLD by regulating its target PPARα and SIRT1.

No MeSH data available.


Related in: MedlinePlus

MiR-34a Target genes prediction, verification and the regulation of miR-34a on PPARα in L02 cells.(A) Seed sequence base-pairing between miR-34a and 3′ UTRs of PPARα as predicted by TargetScan. (B) The three regions of the 3′ UTR of PPARα that were subcloned into the luciferase reporter and mutations of the seed sequences. (C,D) Decreased protein expression of PPARα and SIRT1 were observed in steatosis L02 cells and HFD C57BL/6 liver tissues, and increased in the miR-34a inhibitor treated group in L02 cells and liver tissues. The PPARα and SIRT1 protein expression were detected by western blotting. GAPDH and tubulin were detected as a loading control. The results were standardized to the control group and were presented as the mean ± standard deviation of three independent experiments. The blots were cropped and the gels were run under the same experimental conditions. *P < 0.05.
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f3: MiR-34a Target genes prediction, verification and the regulation of miR-34a on PPARα in L02 cells.(A) Seed sequence base-pairing between miR-34a and 3′ UTRs of PPARα as predicted by TargetScan. (B) The three regions of the 3′ UTR of PPARα that were subcloned into the luciferase reporter and mutations of the seed sequences. (C,D) Decreased protein expression of PPARα and SIRT1 were observed in steatosis L02 cells and HFD C57BL/6 liver tissues, and increased in the miR-34a inhibitor treated group in L02 cells and liver tissues. The PPARα and SIRT1 protein expression were detected by western blotting. GAPDH and tubulin were detected as a loading control. The results were standardized to the control group and were presented as the mean ± standard deviation of three independent experiments. The blots were cropped and the gels were run under the same experimental conditions. *P < 0.05.

Mentions: Two computational methods (TargetScan and PicTar) were used to identify miR-34a targets. Among the hundreds of predicted targets, PPARα is closely associated with lipid metabolism in NAFLD151721 and was chosen for further validation in 293T cells using luciferase reporter assays. Figure 3A shows the predicted miR-34a-binding sites in 3′ UTR of PPARα.


Effect of miR-34a in regulating steatosis by targeting PPARα expression in nonalcoholic fatty liver disease.

Ding J, Li M, Wan X, Jin X, Chen S, Yu C, Li Y - Sci Rep (2015)

MiR-34a Target genes prediction, verification and the regulation of miR-34a on PPARα in L02 cells.(A) Seed sequence base-pairing between miR-34a and 3′ UTRs of PPARα as predicted by TargetScan. (B) The three regions of the 3′ UTR of PPARα that were subcloned into the luciferase reporter and mutations of the seed sequences. (C,D) Decreased protein expression of PPARα and SIRT1 were observed in steatosis L02 cells and HFD C57BL/6 liver tissues, and increased in the miR-34a inhibitor treated group in L02 cells and liver tissues. The PPARα and SIRT1 protein expression were detected by western blotting. GAPDH and tubulin were detected as a loading control. The results were standardized to the control group and were presented as the mean ± standard deviation of three independent experiments. The blots were cropped and the gels were run under the same experimental conditions. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557122&req=5

f3: MiR-34a Target genes prediction, verification and the regulation of miR-34a on PPARα in L02 cells.(A) Seed sequence base-pairing between miR-34a and 3′ UTRs of PPARα as predicted by TargetScan. (B) The three regions of the 3′ UTR of PPARα that were subcloned into the luciferase reporter and mutations of the seed sequences. (C,D) Decreased protein expression of PPARα and SIRT1 were observed in steatosis L02 cells and HFD C57BL/6 liver tissues, and increased in the miR-34a inhibitor treated group in L02 cells and liver tissues. The PPARα and SIRT1 protein expression were detected by western blotting. GAPDH and tubulin were detected as a loading control. The results were standardized to the control group and were presented as the mean ± standard deviation of three independent experiments. The blots were cropped and the gels were run under the same experimental conditions. *P < 0.05.
Mentions: Two computational methods (TargetScan and PicTar) were used to identify miR-34a targets. Among the hundreds of predicted targets, PPARα is closely associated with lipid metabolism in NAFLD151721 and was chosen for further validation in 293T cells using luciferase reporter assays. Figure 3A shows the predicted miR-34a-binding sites in 3′ UTR of PPARα.

Bottom Line: The upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 that are the direct targets of miR-34a.Activation of the central metabolic sensor AMPK was also increased.The miR-34a inhibitor suppressed lipid accumulation and improved the degree of steatosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Hangzhou First People's Hospital, No. 261 Huansha Road, Hangzhou 310006, Zhejiang Province, China.

ABSTRACT
MicroRNA-34a (miR-34a) is thought to be involved in nonalcoholic fatty liver disease (NAFLD). However, the association between altered expression of miR-34a and the pathophysiological features of NAFLD remains unclear. Here, we investigated the mechanisms by which miR-34a influences NAFLD through the PPARα-related pathway. Real-time quantitative PCR, western blotting and other assays kit were used to investigate the expression and function of miR-34a in an NAFLD model. Cultured cells transfected with miR-34a inhibitor and C57BL/6 mice injected with the miR-34a inhibitor through vein tail were conducted for the effects of miR-34a on its target. MiR-34a levels were significantly upregulated in steatosis-induced hepatocytes and in liver tissues of high-fat diet-fed mice. The upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 that are the direct targets of miR-34a. Silencing miR-34a led to an initially increased expression of PPARα, SIRT1 and PPARα's downstream genes. Activation of the central metabolic sensor AMPK was also increased. The miR-34a inhibitor suppressed lipid accumulation and improved the degree of steatosis. Taken together, our data indicated that decreased expression of miR-34a potentially contributes to altered lipid metabolism in NAFLD. Downregulation of miR-34a may be a therapeutic strategy against NAFLD by regulating its target PPARα and SIRT1.

No MeSH data available.


Related in: MedlinePlus