MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism - PubMed (original) (raw)

MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism

Dimitrios Iliopoulos et al. J Lipid Res. 2010 Jun.

Abstract

We previously observed that treatment of mice with a dominant negative form of cJun (dn-cJun) increased the expression of genes involved in lipid metabolism and modulated the expression of nine microRNAs (miR). To investigate the potential effect of these miRs on the expression of the genes of lipid metabolism, we performed studies in cultured HepG2 cells. Transfection of HepG2 cells with sense or antisense miR-370 or miR-122 upregulated and downregulated, respectively, the transcription factor sterol-regulatory element binding protein 1c (SREBP-1c) and the enzymes diacylglycerol acyltransferase-2 (DGAT2), fatty acid synthase (FAS), and acyl-CoA carboxylase 1 (ACC1) that regulate fatty acid and triglyceride biosynthesis. The other seven miRs identified by the miR array screening did not affect the expression of lipogenic genes. miR-370 upregulated the expression of miR-122. Furthermore, the effect of miR-370 on the expression of the lipogenic genes was abolished by antisense miR-122. miR-370 targets the 3' untranslated region (UTR) of Cpt1alpha, and it downregulated the expression of the carnitine palmitoyl transferase 1alpha (Cpt1alpha) gene as well as the rate of beta oxidation. Our data suggest that miR-370 acting via miR-122 may have a causative role in the accumulation of hepatic triglycerides by modulating initially the expression of SREBP-1c, DGAT2, and Cpt1alpha and, subsequently, the expression of other genes that affect lipid metabolism.

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Figures

Fig. 1.

Effect of dn-cJun on the hepatic lipid metabolism and miR gene expression in apoE−/− mice. A: miR changes detected by analysis of a microRNA TaqMan array using hepatic RNA obtained from five dn-cJun-treated mice vs. five control mice. B: Validation of the miR data by real-time SYBR Green PCR assay. C: Real-time PCR analysis of genes involved in fatty acid and triglyceride biosynthesis and catabolism in the liver of dn-cJun-treated vs. control mice. Statistically significant changes in gene expression levels between control and dn-cJun treated mice are indicated by asterisks, P < 0.005. ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c.

Fig. 2.

Modulation of expression of SREBP-1c and genes involved in fatty acid and triglyceride biosynthesis by miR-370 and miR-122 in HepG2 cells. Real-time PCR analysis of SREBP-1c, FAS, ACC1, and DGAT2 after treatment of HepG2 cells with miR-370 at 0, 12, 24, 48, and 72 h post-transfection. Increases in gene expression at all time points were statistically significant (P < 0.005) A: Real-time PCR analysis of SREBP-1c, FAS, ACC1, and DGAT2 after liposomal transfection (50 nM) in HepG2 cells with anti-sense oligonucleotides for inhibition of (B) miR-370; (C) miR-122 and miR-370; and (D) miR-145 (as a control). Statistically significant changes in gene expression at different time points in panels B and C are indicated by * _P_ < 0.005. The symbol + in panels A–C indicates that the difference between 48 h and 72 h post-transfection in FAS and ACC1 mRNA levels were statistically significant (_P_ < 0.05). The difference observed in the expression of FAS and ACC1 between single antisense microRNA (B) or combination of both antisense microRNAs (C) is statistically significant (_P_ < 0.05). There is no statistically significant difference (_P_ > 0.1) in FAS and ACC1 expression levels in as-miR-145 treated HepG2 cells. ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c.

Fig. 3.

Regulation of SREBP-1c and DGAT2 by miR-370 precedes the regulation of FAS and ACC1 genes. A: Real-time PCR analysis of FAS and ACC1 genes after treatment with miR-370 and downregulation of SREBP-1c or DGAT2 or both by treatment with the corresponding siRNA (50 nM) for 48 h. Asterisks indicate statistical significance for both FAS and ACC1 expression levels between untreated and miR-370–treated cells (P < 0.005) as well as miR-370/siscrambled–treated and siSREBP-1c/siDGAT2–treated cells. There is no statistical significance between miR-370 and miR-370/siscrambled–treated cells. B: Real-time PCR analysis of SREBP-1c, FAS, and ACC1 genes after treatment of HepG2 cells with siRNA against SREBP-1c (50 nM) for 48 h. C: Western blot analysis of FAS and ACC1 proteins after treatment of HepG2 cells with siRNA against SREBP-1c (50 nM) for 48 h. D: Real-time PCR analysis of FAS and ACC1 genes after treatment of HepG2 cells with siRNA against DGAT2 (50 nM) for 48 h. E: Western blot analysis of FAS and ACC1 proteins after treatment of HepG2 cells with siRNA against DGAT2 (50 nM) for 48 h. GAPDH protein levels were used as control in both panels C and E. ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c.

Fig. 4.

Effect of miR-370 on the expression of miR-122 in HepG2 cells. A: Real-time PCR analysis of miR-122 following treatment of HepG2 cells with sense and antisense miR-370 or with miR-negative control. B: Real-time PCR analysis of SREBP-1c FAS, ACC1, and DGAT2 gene expression following treatment of HepG2 cells with miR-370 alone or combination of miR-370 and miR-122. Asterisks indicate statistically significant changes between miR control and miR-370–treated HepG2 cells (P < 0.001) and between miR-370/miR control and miR-370/as-miR-122–treated HepG2 cells (P < 0.001). ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c.

Fig. 5.

Effect of miR-370 on Cpt1α expression in HepG2 cells. A: Target sequence in the 3′ UTR of Cpt1α for miR-370. B: Effect of miR-370 on the activity of the SV40 luciferase construct linked to the 3′ UTR of Cpt1α. C: Cpt1α mRNA levels assessed by real-time PCR analysis 24 h and 48 h post-transfection of HepG2 cells with 50 nM miR-370 or miR-122. D: Fatty acid β oxidation levels following transfection of HepG2 cells with 50 μM of miR-370. Statistically significant differences relative to the controls are indicated by asterisks in panels B, C, and D. ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c; UTR, untranslated region.

Fig. 6.

Correlation of hepatic lipid levels with the levels of miR-122 and miR-370 in C57BL/6 mice. A, B: Effect of dn-cJun on the hepatic triglyceride (A) and cholesterol (B) levels, 4 days postinfection with adenoviruses expressing dn-cJun or GFP. C, D: Effect of 2-, 5-, and 8-week high-fat diet on the hepatic triglyceride (C) and cholesterol (D) levels of C57BL/6 mice. (E and F) Effect of 2-, 5-, and 8-week high-fat diets on the hepatic miR-122 (E) and miR-370 (F) levels of C57BL/6 mice. Statistically significant differences relative to the control are indicated by asterisks in panels A–E. G: Strength of miR-gene and gene-gene interactions related with the pathways of fatty acid triglyceride biosynthesis and catabolism. The figure depicts the interaction between miR-122 and miR-370 with their downstream targets. Both miRs indirectly affect initially SREBP-1c and DGAT2. In addition, miR-370 directly and miR-122 indirectly target Cpt1α gene expression. Downstream of the network, SREBP-1c directly and DGAT2 indirectly modulate the expression of FAS and ACC1 mRNA and protein levels. These changes result in increased fatty acid and triglyceride biosynthesis and decreased fatty acid catabolism and are expected to cause hepatic triglyceride accumulation. The line thickness indicates the strength of the correlation. A single arrow indicates direct interaction, while multiple arrows indicate indirect interaction. ACC1, acyl-CoA carboxylase 1; dn-cJun, dominant negative cJun; Cpt1α, carnitine palmitoyl transferase 1α; DGAT2, diacylglycerol acyltransferase-2; miR, microRNA; SREBP-1c, sterol-regulatory element binding protein 1c; UTR, untranslated region.

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MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism - PubMed (original) (raw)