MicroRNA-363 regulation in proliferation and migration of human umbilical vascular smooth muscle cells

Abstract

Objective To investigate microRNA-363 (miR-363) regulation in proliferation and migration of human umbilical vascular smooth muscle cells (VSMCs) and explore the possible mechanisms of miR-363 affecting the biological behavior of VSMCs.Methods Primary cultured human umbilical VSMCs were treated with 30 μg/L of platelet derived growth factor BB (PDGF-BB) to induce phenotypic switch,and miR-363 expression was quantified by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).VSMCs were transfected with miR-363 mimics and miR-NC for negative control,VSMCs proliferation and migration in both groups were evaluated using cell counting kit-8 assays (CCK-8) assay and Transwell migration assay,respectively; phenotypic proteins of α-SMA and Calponin-1 were detected by Western blotting analysis.Comparisons were made between the two groups.Target gene of miR-363 was predicted using bioinformatics analysis,and confirmed by Western blotting analysis.Results VSMCs switched to contractile phenotype 72 hours after they were co-cultured with PDGF-BB.Contractile phenotype protein of α-SMA was down-regulated by 28％ (P ＜0.05) ; the expression of miR-363 was down-regulated by 70％ (P ＜ 0.01).Compared to miR-NC group,significant inhibition of proliferation and migration were observed in miR-363 over-expressed VSMCs,by 32％ and 30％,respectively at 72 hours (P ＜ 0.05).Kruppel-like factor 4 (Klf4),the target gene of miR-363,was down-regulated by 19％ (P ＜ 0.05),52％ (P ＜ 0.01) and 37％ (P ＜ 0.01),respectively 24,48 and 72 hours after VSMCs were transfected with miR-363 ; however,the expression of α-SMA and Calponin-1 were not significant.Conclusion Our results suggest that up-regulation of miR-363 may inhibit proliferation and migration of human VSMCs,and these effects may be achieved partly through targeting the KLF4 expression. Key words: MicroRNA-363 ; Vascular smooth muscle cells ; Proliferation ; Migration; Kruppel-like factor 4