MicroRNA-613 represses lipogenesis in HepG2 cells by downregulating LXRα

Abstract

Background: MicroRNAs (miRNAs) emerge as new important regulators of lipid homeostasis by regulating corresponding genes. MiR-613 is a newly discovered microRNA, of which the biological function is unknown. A recent report has shown that miR-613 downregulates liver X receptor α (LXRα), a ligand-activated nuclear receptor playing an important role in the regulation of lipid metabolism. The purpose of this study is to explore the effect and the molecular basis of miR-613 on lipogenesis in HepG2 cells.

Methods: HepG2 cells were transiently transfected with miR-613 mimic or control microRNA. Real time PCR, Western blot, Luciferase reporter assay and Oil Red O staining were employed to examine the expression of LXRα and its target genes involved in lipogenesis, binding site for miR-613 in 3'-untranslated region (3'-UTR) of LXRα mRNA and lipid droplet accumulation in the cells.

Results: MiR-613 dramatically suppressed the expression of LXRα and its target genes including sterol-regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), carbohydrate responsive element-binding protein (ChREBP) and acetyl-CoA carboxylase (ACC). Reporter assay showed that miR-613 directly bound to 3'-UTR of LXRα mRNA. Moreover, miR-613 significantly repressed LXRα-induced lipid droplet accumulation in HepG2 cells. Ectopic expression of LXRα without 3'-UTR markedly attenuated the miR-613-mediated downregulation of LXRα's target genes and LXRα-induced lipid droplet accumulation.

Conclusions: MiR-613 suppresses lipogenesis by directly targeting LXRα in HepG2 cells, suggesting that miR-613 may serve as a novel target for regulating lipid homeostasis.

Figure 1

MiR-613 decreases LXRα expression at both mRNA and protein levels. A: HepG2 cells were transfected with 80 nM miR-613 mimic or mimic negative control (NC), after 24 hours total protein was subjected to Western blotting analysis and mRNA expression levels of LXRα were analyzed by real-time quantitative PCR and normalized with β-actin control. B and D, HepG2 cells were treated with TO901317 (5 μM) or GW3965 (2 μM) for 24 hours. Western blot analysis for LXRα protein level (B) and Real-time PCR analysis for LXRα mRNA level (D). C and E, 12 hours after transfected with 80 nM miR-613 mimic or NC, HepG2 cells were treated with TO901317 (5 μM) or GW3965 (2 μM) for 24 hours. Western blot analysis for LXRα protein level (C) and Real-time PCR analysis for LXRα mRNA level (E). The relative level of LXRα expression determined using the 2-^△△CT method. *, P < 0.05 (n = 3 for each group).

Figure 2

MiR-613 directly targets LXRα 3^′-UTR. A: A putative binding site of miR-613 in the 3^′-UTR of LXRα mRNA and mutated nucleotide residues are shown in red and italics. B: pMIR/LXRαMIRE or pMIR/LXRαMIRE-mut was cotransfected with 80 nM miR-613 mimic or NC into HepG2 cells. C, pMIR-LXRβ was transiently cotransfected with 80 nM indicated RNA oligonucleotides into HepG2 cells. Luciferase assay was conducted after 24 hours. The data was the firefly luciferase activities normalized with the β-galactosidase activities. *, P < 0.05 (n = 3 for each group).

Figure 3

MiR-613 suppresses LXRα-induced lipogenic genes. A and B: HepG2 cells were treated with TO901317 (5 μM) or GW3965 (2 μM) for 24 hours. Real-time PCR analysis for SREBP-1c, FAS, ChREBP and ACC mRNA level. C and D, after 12 hours transfected with 80 nM miR-613 mimic or NC, HepG2 cells were treated with TO901317 (5 μM) or GW3965 (2 μM) for 24 hours. Real-time PCR analysis for SREBP-1c, FAS, ChREBP and ACC mRNA level. The relative level of lipogenic gene expression determined using the 2-^△△CT method. *, P < 0.05 (n = 3 for each group).

Figure 4

MiR-613 represses lipogenic genes by mediating LXRα. HepG2 cells were cotransfected with 80 nM miR-613 mimic or NC and LXRα-expressing plasmid pEX-LXRα (without 3^′-UTR in LXRα mRNA) for 24 hours. The SREBP-1c, FAS, ChREBP and ACC expression was normalized to β-actin mRNA expression, respectively. The relative level of indicated gene expression determined using the 2-^△△CT method. *, P<0.05 (n=3 for each group).

Figure 5

MiR-613 represses lipogenesis by mediating LXRα. The lipid synthesis was shown by Oil Red O staining (400×). A: After 12 hours transfected with 80 nM miR-613 mimic or NC, HepG2 cells were treated with TO901317 (5 μM) or GW3965 (2 μM) for 24 hours. B: HepG2 cells were cotransfected with 80 nM miR-613 mimic or NC and LXRα-expressing plasmid pEX-LXRα for 24 hours. Results represent 3 independent experiments.