CAJ | 학술논문

目的微小RNA-363(miR-363)对人血管平滑肌细胞(VSMCs)的增殖及迁移的调控,并探讨其影响VSMCs生物学行为的机制.方法 30 μg/L血小板源性生长因子-BB(PDGF-BB)诱导原代培养的人VSMCs表型转化,实时荧光定量聚合酶链反应(FQ-PCR)检测miR-363含量变化.miR-363模拟物及阴性对照(miR-NC)转染VSMCs,细胞计数试剂盒(CCK-8)和Transwell法分别检测细胞增殖、迁移,Western blot法检测表型标志蛋白的表达.生物信息学方法分析miR-363靶基因,Western blot法检测.结果 PDGF-BB刺激72 h后VSMCs向合成型表型改变,表型标志蛋白α-平滑肌肌动蛋白(α-SMA)表达下调28％ (P＜0.05),碱性调宁蛋白(Calponin-1)无显著改变,miR-363的表达量下调70％ (P＜0.01).与miR-NC组比较,在72 h,过表达miR-363组VSMCs增殖抑制32％ (P ＜0.05),迁移抑制30％(P＜0.05);且miR-363的靶基因Kruppel样因子4(KLF4)蛋白表达在转染24、48、72 h后分别降低19％ (P ＜0.05)、52％ (P＜0.01)和37％ (P＜0.01),而其表型标志蛋白α-SMA、Calponin-1表达无明显变化.结论 miR-363可抑制人VSMCs的增殖和迁移,其作用可能是通过靶向KLF4来实现的.
목적 미소RNA-363(miR-363)대인혈관평활기세포(VSMCs)적증식급천이적조공,병탐토기영향VSMCs생물학행위적궤제.방법 30 μg/L혈소판원성생장인자-BB(PDGF-BB)유도원대배양적인VSMCs표형전화,실시형광정량취합매련반응(FQ-PCR)검측miR-363함량변화.miR-363모의물급음성대조(miR-NC)전염VSMCs,세포계수시제합(CCK-8)화Transwell법분별검측세포증식、천이,Western blot법검측표형표지단백적표체.생물신식학방법분석miR-363파기인,Western blot법검측.결과 PDGF-BB자격72 h후VSMCs향합성형표형개변,표형표지단백α-평활기기동단백(α-SMA)표체하조28％ (P＜0.05),감성조저단백(Calponin-1)무현저개변,miR-363적표체량하조70％ (P＜0.01).여miR-NC조비교,재72 h,과표체miR-363조VSMCs증식억제32％ (P ＜0.05),천이억제30％(P＜0.05);차miR-363적파기인Kruppel양인자4(KLF4)단백표체재전염24、48、72 h후분별강저19％ (P ＜0.05)、52％ (P＜0.01)화37％ (P＜0.01),이기표형표지단백α-SMA、Calponin-1표체무명현변화.결론 miR-363가억제인VSMCs적증식화천이,기작용가능시통과파향KLF4래실현적.
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.