MicroRNA-133a regulates insulin-like growth factor-1 receptor expression and vascular smooth muscle cell proliferation in murine atherosclerosis

Abstract

Objective: MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis.

Methods and results: Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE(-/-)) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE(-/-) aortas. ApoE(-/-) VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3'UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE(-/-) VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE(-/-) VSMC than in WT cells.

Conclusion: MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1-stimulated VSMC survival and growth.

Keywords: Artery; Atherosclerosis; Insulin-like growth factor; MicroRNA; Smooth muscle cell.

Fig. 1. Expression of miR-1, miR-133a and IGF-1R in WT and ApoE ^−/− aortic smooth muscle cells

(A) qRT-PCR analysis of miR-1 and miR-133a in total RNA extracted from VSMC of wild type (WT) and ApoE^−/− mice. (B) qRT-PCR analysis of IGF-1R mRNA in WT and ApoE ^−/− VSMC. (C) Western blot analysis of IGF-1R (upper panel) and GAPDH (lower panel) proteins in WT and ApoE^−/− mice. (D) Densitometry of protein bands in blots. Data represent means±SD. * p < 0.05.

Fig. 2. MiR-133a regulation of IGF-1-stimulated growth of WT and ApoE ^−/− aortic smooth muscle cells

(A) Analysis of VSMC growth after treated with miR-133a inhibitors. EdU incorporation was analyzed in synchronized VSMC cultured in DME medium supplemented with 10% LPDS and treated with miR-133a inhibitor or precursor in the presence or absence of IGF-1 for different time points. (B) WT VSMC treated with miR-133a inhibitor and/or IGF-1. (C) ApoE^−/− VSMC with miR-133a inhibitor and/or IGF-1. (D) WT VSMC with miR-133a precursor and/or IGF-1. (E) ApoE^−/− VSMC with miR-133a precursor and/or IGF-1. Data represent means ± SD. *p < 0.05.

Fig. 3. MiR-133a regulation of α-SM-actin expression in aortic smooth muscle cells isolated from WT and ApoE ^−/− mice

(A) α-SM-actin (upper panel) and GAPDH (lower panel) Western blot. (B) Densitometry of α-SM-actin protein bands in the blots normalized by that of GAPDH. (C) WT VSMC treated with scrambled miRNA control. (D) WT VSMC treated with miR-133a inhibitor. (E) ApoE^−/− VSMC treated with scrambled miRNA control. (F) ApoE^−/− VSMC treated with miR-133a inhibitor. (G) Quantification of α-SM-actin positive cells in cultures treated with scrambled controls or miR-133a inhibitor. The positive cells were identified by using an image analysis computer software with the non-immune IgG-stain fluorescence as the threshold. Data represent means ± SD. *p < 0.05. Images taken using 20× objective.

Fig. 4. Impacts of miR-133a regulators on expression of IGF-1R protein and miR-133a in aortic smooth muscle cells

(A and B) Western blot analysis of IGF-1R and GAPDH. (C and D) IGF-1R expression quantified by densitometry. (E and F) miR-133a expression detected by qRT-PCR. Data represent means ± SD. *p < 0.05.

Fig. 5. Luciferase reporter assays and IGF-1R mRNA half-life detection

(A) Schematic representation of psiCHECK2 luciferase reporter constructs. The IGF-1R mRNA 3’UTR sequence containing miR-133a targeting site (IGF-1R-3’UTR) and miR-133a anti-sense (miR-133a AS) were inserted into the psiCHECK2 reporter vector at the site downstream of the luciferase reporter gene. (B) Interaction between miR-133a and IGF-1R 3’UTR sequence were analyzed by luciferase reporter assays. VSMC cells were transfected with psiCHECK2 luciferase reporter vectors containing miR-133a targeting site downstream of the Renilla luciferase gene (50 ng), and the internal Firefly luciferase gene was used to normalize for transfection efficiency. A pLVX-miR-133a expression vector was co-transfected (150 ng). Dual-luciferase assays were performed 48 hours after transfection. (C and D) MiR-133a precursors increase, while miR-133a inhibitors reduce, the half-life of IGF-1R mRNA in aortic smooth muscle cells. Total RNA was extracted from synchronized VSMC. The mRNA stability was denoted with the IGF-1R mRNA decay curve after 5 µg /mL Act-D treatment. Synchronized VSMC from WT and ApoE^−/− mice were treated with miR-133a inhibitor or precursor in the presence of Act-D at different time points. At the end of culture, cellular RNA was extracted. IGF-1R mRNA was measured at each time point using qRT-PCR. Data represent means ± SD. *p < 0.05.