MicroRNA Expression Signature and Antisense-Mediated Depletion Reveal an Essential Role of MicroRNA in Vascular Neointimal Lesion Formation

Abstract

Abstract-MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21-mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke. (Circ Res. 2007;100:1579-1588.)Key Words: microRNAs Ⅲ vascular smooth muscle cells Ⅲ proliferation Ⅲ apoptosis Ⅲ neointimal formation M icroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs of Ϸ22 nucleotides and constitute a novel class of gene regulators. [1][2][3] Although the first miRNA, lin-4, was discovered in 1993, 4,5 their presence in vertebrates was confirmed only in 2001. 6 MiRNAs are initially transcribed by RNA polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts. 7 The primiRNAs are processed by the RNase III enzymes, Drosha and Dicer, to generate 18-to 24-nucleotide mature miRNAs. The mature miRNAs negatively regulate gene expression in 1 of 2 ways that depend on the degree of complementarity between the miRNA and its target. MiRNAs that bind to 3ЈUTR of mRNA with imperfect complementarity block protein translation. In contrast, miRNAs that bind to mRNA with perfect complementarity induce targeted mRNA cleavage. Currently, more than 400 miRNAs have been cloned and sequenced in human, and the estimated number of miRNA genes is as high as 1000 in the human genome. 8,9 As a group, miRNAs are estimated to regulate 30% genes of the human genome. 10 Analogous to the first RNA revolution in the 1980s with Cech discovering the enzymatic activity of RNA, 11 this recent discovery of RNAi and miRNA may represent the second RNA revolution. 12 Small interfering RNAs (siRNAs) are another class of small noncoding RNAs that have similar mechanism for gene expression regulation. However, they are different from each other. 5,13 The chief difference lies in their origins....

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“…miR-195 was reported to be up-regulated in cardiac hypertrophy, and transgenic mice overexpressing miR-195 resulted in cardiac growth with disorganization of cardiomyocytes [35]. Ji et al [36] reported that miR-195 was highly expressed in rat carotid artery and down-regulated in the process of wound healing caused by balloon injury (see its supplemental data). The Neurogenin3 transcript is targeted by miR-195, which contributes to islet cell regeneration in pancreas [37].…”

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“…This team identified p27 (Kip1) and p57 (Kip2) as potential targets for these miRNAs in these cells. Further by knocking down miR-221 and miR-222 in rat carotid arteries, they revealed therapeutic potential for these miRNAs in suppressing cell proliferation in vivo and neointimal lesion formation after angioplasty [149,150]. The role of miRNAs in regulating endothelial cells in response to hypoxia was also studied [151].…”

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“…In keeping with their roles in cell proliferation, miRs are key participants in neointimal development. Arterial injury leads to rapid differential expression in miRs, with up-regulation of miR-21, miR-146, miR-214 and miR-352, and down-regulation of miR-125a, miR-125b, miR-133a, miR-145, miR-143, miR-347 and miR-365 [71]. Antisense knockdown of the most up-regulated of these (miR-21) reduces neointima, again suggesting that manipulation of single miRs has the potential for therapeutic benefit.…”