MicroRNA let-7g alleviates atherosclerosis via the targeting of LOX-1 in vitro and in vivo

Abstract

Atherosclerosis is a chronic arterial disease and the leading cause of stroke and myocardial infarction. Micro-RNAs (miRNAs or miRs) have been reported to act as essential modulators during the progression of atherosclerosis. Although miR-let-7g has been demonstrated to contribute to maintaining endothelial function and vascular homeostasis, it is not known whether miR-let-7g exerts a therapeutic effect on experimental atherosclerosis. The aim of this study was to investigate the effects of miR-let-7g on atherosclerosis in vivo and in vitro and to explore its underlying mechanisms. Data from our study showed that exogenous lectin‑like oxidized low‑density lipoprotein receptor‑1 (LOX-1 or OLR1) overexpression resulted in the significant promotion of proliferation and migration of human aortic smooth muscle cells (ASMCs), whereas such changes induced by LOX-1 were obviously suppressed by transfection of miR‑let‑7g. We later confirmed that LOX-1 is a potential target of miR-let-7g, and miR-let-7g markedly inhibited LOX-1 expression in ASMCs by directly binding to the 3' untranslated region of LOX-1. Furthermore, in a hyperlipidemic apolipoprotein E knockout (ApoE-/-) mouse model, intravenous delivery of miR-let-7g mimics obviously attenuated high-fat diet-induced neointima formation and atherosclerotic lesions, accompanied by the significant downregulation of LOX-1, which was consistent with the effect of miR-let-7g on ASMCs. Taken together, our data revealed that miR-let-7g exhibits anti-atherosclerotic activity, at least partially by targeting the LOX-1 signaling pathway. This study suggests that miR-let-7g may be a therapeutic candidate for treating atherosclerosis, and provides novel insight into miRNA-based therapy for this disease.

Figure 1

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) overexpression stimulates aortic smooth muscle cell (ASMC) proliferation and migration. ASMCs were transfected with pCMV3-LOX-1 to induce LOX-1 overexpression or pCMV3 vector as a negative control. (A) Cell proliferation was determined using MTT assay at 0, 24, 48, 72 and 96 h post-tranfection. (B) Cell migration was detected using a wound healing assay and (D) Transwell migration assay, and (C) the cell migration rate of these two methods was respectively analyzed as described in Materials and methods. Original magnification was ×100 for (B) and ×200 for (D). Data are presented as mean ± SD (n=3). ^**p<0.01 vs. the control; ^##p<0.01 vs. the pCMV3 vector group.

Figure 2

miR-let-7g attenuates lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)-induced aortic smooth muscle cell (ASMC) proliferation and migration. ASMCs were co-transfected with pCMV3-LOX-1 or pCMV3 vector and pcDNA3.1-let-7g or pcDNA3.1 vector for overexpression of LOX-1 or let-7g or both. (A) Cell proliferation was determined using the MTT assay at 0, 24, 48, 72 and 96 h post-tranfection. (C) Cell migration was detected using a wound healing assay and (D) Transwell migration assay, and (B) the ratio of cell migration was analyzed. Original magnification was ×100 for (C) and ×200 for (D). Data are presented as mean ± SD (n=3). ^*p< 0.05, ^**p<0.01 vs. pCMV3+pcDNA3.1; ^#p<0.05, ^##p<0.01 vs. pCMV3-LOX-1+pcDNA3.1.

Figure 3

miR-let-7g inhibits lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression in human aortic smooth muscle cells (ASMCs) by binding to its 3′ untranslated region (3′UTR). (A) A sequence complementary to the seed sequence of let-7g was observed in the 3′UTR of LOX-1 mRNA in the human (Hsa-) and mouse (Mmu-). (B) ASMCs were co-transfected with pmirGLO-LOX-1-3′UTR luciferase reporter plasmid and pcDNA3.1-let-7g, mutant or pcDNA3.1 plasmid. At 24 h post-transfection, luciferase assay was performed. A reporter containing not LOX-1-3′UTR but pmirGLO was used as a negative control. (C and D) ASMCs were transfected with pcDNA3.1-let-7g or its control plasmid, and then (C) the expression levels of LOX-1 mRNA and (D) LOX-1 protein were determined using real-time PCR and western blot analysis, respectively. β-actin served as the internal control. Data are presented as mean ± SD (n=3). ^**p<0.01 vs. the control; ^##p<0.01 vs. the pcDNA3.1.

Figure 4

miR-let-7g alleviates atherosclerotic lesions in apolipoprotein E (ApoE^−/−) mice. (A) Aortic tissue sections from each group were stained with hematoxylin and eosin (H&E) for histological assessment. Representative images are shown. Original magnification was ×400. (B) Lumen area, (C) intima area and (D) medial area of the aortas from each group were analyzed by Image-Pro Plus 6.0. (E) The ratio of intima area to medial area was calculated. Data are presented as mean ± SD (n=6). ^**p<0.01 vs. the control group; ^##p<0.01 vs. the model group; ^§p<0.05 and ^§§p<0.01 vs. the let-7g NC group.

Figure 5

miR-let-7g downregulates the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the aortas of apolipoprotein E knockout (ApoE^−/−) mice. (A) The expression levels of LOX-1 mRNA and (B) LOX-1 protein in the arteries of C57BL/6 mice and ApoE^−/− mice were evaluated using real-time PCR and western blot analysis, respectively. β-actin was used as the internal control. Data are presented as mean ± SD (n=6). ^**p<0.01 vs. the control group; ^##p<0.01 vs. the model group; ^§§p<0.01 vs. the let-7g NC group. (C) Immunofluorescence staining was performed to detect the protein expression of LOX-1 (red) in the arterial sections from each group. Nuclei were visualized by DAPI staining (blue). The representative images of each group are shown. Original magnification was ×400.