A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia

Abstract

MicroRNAs (miRNAs) comprise a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression. Functionally, an individual miRNA is as important as a transcription factor because it is able to regulate the expression of its multiple target genes. Recently, miR-221 and miR-222 have been found to play a critical role in cancer cell proliferation. However, their roles in vascular smooth muscle cell (VSMC) biology are currently unknown. In the present study, the time course changes and cellular distribution of miR-221 and miR-222 expression were identified in rat carotid arteries after angioplasty, in which their expression was upregulated and localized in VSMCs in the injured vascular walls. In cultured VSMCs, miR-221 and miR-222 expression was increased by growth stimulators. Knockdown of miR-221 and miR-222 resulted in decreased VSMC proliferation in vitro. Using both gain-of-function and loss-of-function approaches, we found that p27(Kip1) and p57(Kip2) were 2 target genes that were involved in miR-221- and miR-222-mediated effect on VSMC growth. Finally, knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and neointimal lesion formation after angioplasty. The results indicate that miR-221 and miR-222 are novel regulators for VSMC proliferation and neointimal hyperplasia. These findings may also represent promising therapeutic targets in proliferative vascular diseases.

Fig. 1. Expression and distribution of miR-221 and miR-222 in balloon-injured rat carotid arteries

(A). The time course changes of miR-221 and miR-222 expression determined by qRT-PCR. Note: n=6; *P<0.05 compared with uninjured control. (B). Representative Masson's trichrome staining. (C) Negative control (no SM α-actin antibody, no miRNA probe) for In situ hybridization and immunofluorescence. (D) Scrambled probe control 1 (no SM α-actin antibody, but had scrambled miRNA probe). (E) Scrambled probe control 2 (had SM α-actin antibody and scrambled miRNA probe). (F) In situ hybridization of miR-221 (dot green color), immunofluorescence of smooth muscle cell marker SM α–actin (red color) and cell nuclear staining by DAPI (blue color). (G) In situ hybridization of miR-222 (dot green color), immunofluorescence of smooth muscle cell marker SM α–actin (red color) and cell nuclear staining by DAPI (blue color). Note: Autofluorescence in the elastic laminae is demonstrated as green color, but is not dot green (C–G).

Fig. 2. The effect of platelet-derived growth factor (PDGF) and serum on the expression of miR-221 and miR-222 in cultured rat VSMCs

(A) PDGF-BB (20 ng/ml) caused a time-dependent increase in miR-221 and miR-222 expression as demonstrated by qRT-PCR. (B) Serum (10%) caused a time-dependent increase in miR-221 and miR-222 expression as demonstrated by qRT-PCR. (C) PDGF-BB caused a dose-dependent increase in miR-221 and miR-222 expression in cultured VSMCs at 48 h after treatment. Note: n=6; *P<0.05 compared with 0 h groups in Fig. 2A & B, and with vehicle control (0) in Fig. 2C.

Fig. 3. The effect of miR-221 and miR-222 inhibitor on VSMC proliferation in vitro

(A) Knocking down of miR-221 and miR-222 expression by their inhibitors, 2'OMe-miR-221 (100 nM), 2'OMe-miR-222 (100 nM), and 2'OMe-miR-221 plus 2'OMe-miR-222 (100 nM). (B) 2'OMe-miR-222 (100 nM) decreased cell numbers and (C) BrdU incorporation at 48 h after culture with DMEM containing 10% FBS. (D) Representative BrdU-stained cell photomicrographs (top panel), their corresponding total cell photomicrographs stained by DAPI (medial panel) and merged photomicrographs (bottom panel). Note: n=8; *P<0.05 compared with vehicle control.

Fig. 4. p27(Kip1) and p57(Kip2) are target genes of miR-221 and miR-222 in cultured VSMCs

(A) p27(Kip1) and p57(Kip2) were downregulated in proliferative VSMCs stimulated with PDGF-BB (20 ng/ml). Note: Top panel was the representative western blot and bottom panel was the quantification of p27(Kip1) and p57(Kip2) protein. n=3; *P<0.05 compared with 0 h group. (B) Treatment with 2'OMe-miR-222 for 48 h decreased the expression of miR-221 and miR-222 in cultured VSMCs. In contrast, control oligo or unrelated miRNA inhibitor had no effect on miR-221 and miR-222 expression. Note: n=3; *P<0.05 compared with vehicle group. (C) Ad-miR-221 and Ad-miR-222 increased the expression of miR-221 and miR-222 in cultured VSMCs. In contrast, control adenovirus, Ad-GFP or unrelated adenovirus control, Ad-miR-31 had no effect on the expression of miR-221 and miR-222. Note: n=6; *P<0.05 compared with vehicle (blank) group. (D) p27(Kip1) and p57(Kip2) protein levels were upregulated by 2'OMe-miR-222. Note: Top panel was the representative western blot and bottom panel was the quantification of p27(Kip1) and p57(Kip2) protein. n=3; *P<0.05 compared with vehicle group. (E) p27(Kip1) and p57(Kip2) protein levels were down regulated by Ad-miR-221 or Ad-miR-222. Note: Top panel was the representative western blot and bottom panel was the quantification of p27(Kip1) and p57(Kip2) protein. Note: n=3; *P<0.05 compared with vehicle (blank) group. (F). The effects of miR-221 or miR-222 overexpression by Ad-miR-221 or Ad-miR-222 on mRNA expression of p27(Kip1) and p57(Kip2). Note: n=3; *P<0.05 compared with vehicle (blank) group.

Fig. 5. miR-221 and miR-222 are able to directly bind to p27(Kip1) and p57(Kip2) and inhibit their expression in HEK 293 cells

A construct in which a fragment of the 3’-UTR of either p27(Kip1) or p57(Kip2) mRNA containing the putative miR-221 and miR-222 binding sequences was cloned into a firefly luciferase reporter construct and transfected into HEK 293 cells with either vehicle (vehicle), an empty plasmid (pDNR-CMV), a plasmid expressing miR-221 (pmiR-221), miR-222 (pmiR-222), or a control plasmid expressing an unrelated miRNA, miR-145 (pmiR-145). The constructs with mutated fragment of the 3’-UTR of either p27(Kip1) or p57(Kip2) mRNA without the putative miR-221 and miR-222 binding sequences were used as mutated controls. (A) pmiR-221 and pmiR-222, but not pmiR-145 and pDNR-CMV, increased miR-221 or miR-222 expression in HEK 293 cells. (B) pmiR-221 and pmiR-222, but not pDNR-CMV or pmiR-145 inhibited luciferase activity. In the mutated control groups, the inhibitory effect of pmiR-221 and pmiR-222 was disappear. Note; N=5; *P<0.05 compared with vehicle control.

Fig. 6. miR-221 and miR-222-mediated effect on VSMC proliferation is decreased in p27(Kip1) and p57(Kip2) deficient cells

(A) Representative western blot of p27(Kip1) and p57(Kip2) protein. (B) p27(Kip1) and p57(Kip2) expression at protein level was depleted by their siRNAs (25 nM). Note: n=6; *P<0.05 compared with vehicle control. (C) miR-221 and miR-222 inhibitor, 2'OMe-miR-222-mediated inhibitory effect on VSMC proliferation was decreased in p27(Kip1) and p57(Kip2) depleted cells. Note: The relative inhibition of cells without 2'OMe-miR-222 was defined as 0%, while in 2'OMe-miR-222 plus vehicle-treated cells was defined as 100% inhibition. N=8; *P<0.05 compared with vehicle control.

Fig. 7. p27(Kip1) and p57(Kip2) are target genes of miR-221 and miR-222 in the vascular walls after angioplasty

A. Both p27(Kip1) and p57(Kip2) proteins were downregulated in balloon-injured rat carotid arteries. (B) miR-221 and miR-222 were downregulated by 2'OMe-miR-222 in balloon-injured vascular walls. (C) Representative western blot of p27(Kip1) and p57(Kip2) protein in the injured vascular walls treated with vehicle, control oligo or 2'OMe-miR-222. (D) In 2'OMe-miR-222-treated vessels, expression of p27(Kip1) and p57(Kip2) was upregulated. Note; N=6; *P<0.05 compared with vehicle control.

Fig. 8. Downregulation of miR-221 and miR-222 decreases cell proliferation and neointima formation in rat carotid artery after angioplasty

(A) Representative immunofluorescence of PCNA in rat carotid arteries at 14 days after balloon-injury. Note: Green color was the immunofluorescence of PCNA that represented proliferating cells. Blue was cell nuclear staining by DAPI that reflects total cells. Red color was the autofluorescence in elastic laminae. (B) Quantification of the proliferative cells showed that, compared with vehicle-treated and control oligo-treated vessels, fewer cells were proliferating in the injured vascular walls treated with 2'OMe-miR-222. (C) Downregulation of the miR-221 and miR-222 by 2'OMe-miR-222 decreased neintimal formation. (D) Representative Masson's trichrome stained photomicrographs of rat carotid arteries from different groups. Note; N=8; *P<0.05 compared with vehicle control.