miR-203 Inhibits Alcohol-Induced Hepatic Steatosis by Targeting Lipin1

Abstract

Alcoholic liver disease (ALD) is a global liver disease which characterized by liver inflammation, fatty liver, alcoholic hepatitis, or liver cirrhosis. Alcohol abuse is one of the main reasons for liver disease. Alcoholic fatty liver (AFL) disease is the early stage of ALD and associated with the excessive lipids accumulation in hepatocytes as well as oxidative stress. MicroRNA-203 (miR-203) is known to suppress the proliferation and metastasis of hepatocellular carcinoma, but the role in the progression of alcoholic liver disease is not clear and is warranted for further investigation. In the present study, we have found the expression of miR-203 is down-regulated in Gao-Binge alcoholic mice model and ethanol-induced AML-12 cell lines in vitro. Furthermore, over-expression of miR-203 decrease the lipids accumulation in liver and ethanol-induced AML-12 cells. Mechanistically, we identified that Lipin1 is a key regulator of hepatic lipid metabolism, and acts as a downstream target for miR-203. In summary, our results suggested that over-expression of miR-203 inhibited the liver lipids accumulation and the progression of AFL by targeting Lipin1.

Keywords: Gao-binge; Lipin1; alcoholic fatty liver; lipid metabolism; miR-203.

FIGURE 1

miR-203 was down-regulated in EtOH-fed mice and EtOH-induced AML-12 cells. (A) Representative hematoxylin and eosin (H&E) staining of liver tissues (×400). (B) Body weights and the liver to body weight ratio after ethanol feeding. (C) Hepatic triglyceride (TG) and total cholesterol (TCH) levels. (D) Serum ALT and AST levels. (E) One-step qRT-PCR for miR-203 expression in EtOH-fed mice liver tissues compared to normal liver tissues and AML-12 cell line. (F) AML-12 cell line was treated with ethanol (0, 50, 75, 100, 150, 200) mM for 24 h the cell viability. ^∗p < 0.05, ^∗∗p < 0.01 versus control group.

FIGURE 2

miR-203 inhibited liver lipids accumulation in vivo. (A) Lenti-mir-203 and Lenti-NC expression in mice liver. (B) Expression of miR-203 was confirmed by qRT-PCR. (C) The liver tissues H&E and Oil Red O staining (x100). (D) Liver triglyceride (TG) levels. (E,F) qRT-PCR and Western blot analysis for mRNA and protein expression of lipid metabolism markers PPAR-α^∗ SREBP-1, inflammation markers IL-6, TNF-α. ^∗p < 0.05, ^∗∗p < 0.01 versus control group. ^#p < 0.05, ^##p < 0.01 versus NC group.

FIGURE 3

miR-203 inhibited hepatocyte lipids accumulation in vitro. (A) Transfection effect of miR-203 mimics was confirmed by qRT-PCR. (B) The cellular Oil Red staining (x200). (C) The cellular TG and TCH levels. (D,E) qRT-PCR and “Western blot” analysis for mRNA and protein expression of lipid metabolism markers PPAR-α^∗ SREBP-1, inflammation markers IL-6, TNP-α. ^∗p < 0.05, ^∗∗p < 0.01 versus control group. ^#p < 0.05, ^##p < 0.01 versus NC group.

FIGURE 4

miR-203 directly regulated the expression of Lipin1. (A) Bioinformatics analyses show the seed sequence of miR-203 bind to the 3′-UTR of LPIN1 mRNA. (B) “Wild type” 3′-UTR of LPIN1 gene was cloned into the firefly and Renilla reporter plasmid. The LPIN1-3′ UTR constructs or blank plasmid were transfected into AML-12 cells with control or miR-203 mimics, followed by dual luciferase assays. (C,D) Western blot analysis for protein expression of LPIN1 after transfected with miR-203 mimics or miR-NC, mir-203 inhibitor or inhibitor-NC. ^∗p < 0.05, ^∗∗p < 0.01 versus control group. ^#p < 0.05, ^##p < 0.01 versus NC group.

FIGURE 5

Knocked down Lipinl contributed to inhibit hepatocyte lipids accumulation in vitro. (A) IHC analysis on liver tissues between the four groups. (B) Western blot analysis for Lipinl expression on mice liver tissues. (C) AML-12 cells were transfected with miR-203 mimic or miR-NC and then stimulated with ethanol 100 mM 24 h, Western analysis for Lipinl protein expression. (D) AML-12 cells were transfected with Lipinl-siRNA or control siRNA and then stimulated with ethanol 100 mM 24 h, Western analysis for Lipinl protein expression. (E) Western analysis for PPAR-α, SREBP-1, FASN protein expression after transfection. ^∗p < 0.05, ^∗∗p < 0.01 versus control group. ^#p < 0.05, ^##p < 0.01 versus NC group.

FIGURE 6

miE-203 inhibited cytoplasmic localization of Lipin1 in mouse AML-12 cells. (A) Representative photomicrographs of immunofluorescence Lipin1 (green) or DAPI (blue) m AML-12 cells transfected with miR-203 mimics or NC and then treated with ethanol (100 mM) for 24 h (x200). (B,C) Representative western blotting analysis of the Lipin1 protein expression levels in cytoplasm (Cyto) or nucleus (Nuc).