Identification of microRNA that represses IRS-1 expression in liver

Abstract

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression and have been shown to participate in almost every cellular process. Several miRNAs have recently been implicated in glucose metabolism, but the roles of miRNAs in insulin-resistant conditions, such as obesity or type 2 diabetes, are largely unknown. Herein, we focused on miR-222, the expression of which was increased in the livers of high fat/high sucrose diet-fed mice injected with gold thioglucose (G+HFHSD). Overexpression of miR-222 in primary mouse hepatocytes attenuated Akt phosphorylation induced by insulin, indicating that miR-222 negatively regulates insulin signaling. As per in silico analysis, miR-222 potentially binds to the 3' untranslated region (3' UTR) of the IRS-1 gene, a key insulin signaling molecule. In fact, IRS-1 protein expression was decreased in the livers of G+HFHSD-fed mice. We further confirmed a direct interaction between miR-222 and the 3' UTR of IRS-1 via luciferase assays. Our findings suggest that up-regulation of miR-222 followed by reduction in IRS-1 expression may be a viable mechanism of insulin resistance in the liver.

Fig 1. miR-222 expression is up-regulated in the livers of G+HFHSD-fed mice.

(A) Body weight was measured during each treatment in NC or G+HFHSD-fed mice (n = 7–8 per group). (B, C) Fasting blood glucose and insulin levels were measured after 24 weeks of treatment. (D) HOMA-IR was calculated using fasting blood glucose and insulin levels after 24 weeks of treatment. (E, F) After 24 weeks of treatment, microRNA was collected from the livers of these mice. miR-222 and miR-221 expression were analyzed by qRT-PCR (n = 8 per group). (G) The proteins in the livers of these mice were analyzed with WB. The IRS-1 and IRS-2 levels were quantified by normalization with β-actin. (n = 4 per group). (H) Irs-1 mRNA expression in the livers of these mice were analyzed by qRT-PCR. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 compared with NC-fed mice.

Fig 2. Effect of miR-222 overexpression on insulin signaling in primary mouse hepatocytes.

(A) Primary hepatocytes were transfected with 30 nM negative control oligos (control) or miR-222 mimic using HilyMax. After 2 days of transfection, miR-222 overexpression in the cells was confirmed by qRT-PCR (n = 6 per group). (B) Cells overexpressing miR-222 were treated with insulin (100 nM) for 10 min. Cells were harvested, and protein levels involved in insulin signaling were determined by WB. The IRS-1 and P-Akt levels were quantified by normalization with β-actin and total Akt. The values are expressed as mean ± SD from 4 independent experiments. (C) Irs-1 and Irs-2 mRNA expression in the cells overexpressing miR-222 were analyzed by qRT-PCR. (D) Each mRNA involved in insulin signaling was analyzed in the cells overexpressing miR-222 (n = 10 per group). Data are presented as means ± SD. *p < 0.05, **p < 0.01 compared with the control group.

Fig 3. Target site of miR-222 in the 3' UTR of mouse Irs-1 and assessment of its binding.

(A) The seed sequence of miR-222 and the sequence of 3' UTR of Irs-1. (B) A pmirGLO-based 3′ UTR reporter vector consisting of luciferase cDNA followed by the 3' UTR of murine Irs-1 mRNA (WT or Mut). (C) HEK-293 cells were co-transfected with the luciferase reporter construct containing WT or Mut 3' UTR of mouse Irs-1 and the miR-222 mimic or the negative control oligos. After 2 days of treatment, a dual-luciferase assay of these cells was measured (n = 6 per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01 compared with cells transfected with the negative control group. MRE: microRNA response element; UTR: untranslated region; CDS: coding sequence.

Fig 4. Effect of miR-222 overexpression in HuH-7 cells.

(A) HuH-7 cells were transfected with 30 nM of the negative control or miR-222 mimic. After 2 days of transfection, miR-222 overexpression was confirmed by qRT-PCR (n = 6 per group). (B) Cells overexpressing miR-222 were treated with insulin (100 nM) for 10 min. Cells were harvested, and protein levels involved in insulin signaling were determined with WB. The IRS-1 and P-Akt levels were quantified by normalization with β-actin and total Akt. The values are expressed as the mean ± SD from 4 independent experiments. (C) The pmirGLO-based 3' UTR reporter vector consisted of luciferase cDNA followed by the 3' UTR of the WT or Mut human IRS-1 mRNA. (D) HEK-293 cells were co-transfected with the luciferase reporter construct containing WT or Mut 3' UTR of human IRS-1 and the miR-222 mimic or the negative control oligos. After 2 days of treatment, a dual-luciferase assay of these cells was measured (n = 10 per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01 compared with the control group.