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MiR-122 regulates collagen production via targeting hepatic stellate cells and suppressing P4HA1 expression.

ABSTRACT: MicroRNAs (miRNAs) have been shown to be involved in many biological processes by affecting their target gene expression. miR-122 has been extensively studied in hepatocarcinogenesis. However, the role of miR-122 in liver fibrosis remains unknown.The mRNA expression levels of miR-122, prolyl 4-hydroxylase subunit alpha-1 (P4HA1), and CCAAT/enhancer binding protein alpha (C/EBP?) were assessed by real-time PCR. The protein expression levels of P4HA1, C/EBP? and collagen, type I, alpha 1 (COL1A1) were analyzed by Western blot and immunofluorescence. MTT assay was used to assess cell proliferation. Chromatin immunoprecipitation (ChIP) assay was used to examine the binding activity of C/EBP? to miR-122 promoter.miR-122 expression was significantly reduced in transactivated HSCs and in the livers of mice treated with CCl(4). Overexpression of miR-122 inhibited the proliferation of LX2 cells. We also demonstrated that P4HA1 was a target gene of miR-122. The mRNA expression level of PAHA1 inversely correlated with that of miR-122 in HSCs and in the mouse liver. Overexpression of miR-122 markedly attenuated the expression of P4HA1 via targeting a binding site located at 3'-UTR of P4HA1 mRNA. We further showed that miR-122 overexpression led to decreased collagen maturation and ECM production. Finally, the binding activity of C/EBP? to miR-122 promoter was significantly decreased in activated HSCs.Our study suggests that miR-122 may play an important role in negatively regulating collagen production in HSCs and that targeted expression of miR-122 in HSCs may represent a new strategy for the treatment of liver fibrosis.

SUBMITTER: Li J

PROVIDER: S-EPMC3619187 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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miR-122 regulates collagen production via targeting hepatic stellate cells and suppressing P4HA1 expression.

Li Jiang J Ghazwani Mohammed M Zhang Yifei Y Lu Jianqin J Li Jilong J Fan Jie J Gandhi Chandrashekhar R CR Li Song S

Journal of hepatology 20121121 3

<h4>Background & aims</h4>MicroRNAs (miRNAs) have been shown to be involved in many biological processes by affecting their target gene expression. miR-122 has been extensively studied in hepatocarcinogenesis. However, the role of miR-122 in liver fibrosis remains unknown.<h4>Methods</h4>The mRNA expression levels of miR-122, prolyl 4-hydroxylase subunit alpha-1 (P4HA1), and CCAAT/enhancer binding protein alpha (C/EBPα) were assessed by real-time PCR. The protein expression levels of P4HA1, C/EB ...[more]

PMID: 23178710

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Project description:OBJECTIVE:It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies. METHODS:Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-13C5,15N2]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples. RESULTS:The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of 13C- and/or 15N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased 13C-Gln, 13C-á-ketoglutarate, 13C-isocitrate, and 13C-citrate levels. In contrast, 13C-phosphoenolpyruvate and 13C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins. CONCLUSIONS:Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.

Dataset Information

MiR-122 regulates collagen production via targeting hepatic stellate cells and suppressing P4HA1 expression.

Publications

miR-122 regulates collagen production via targeting hepatic stellate cells and suppressing P4HA1 expression.

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