摘要
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30μg/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一363靶基因，Western blot法检测。结果PDGF-BB刺激72h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72h，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、72h后分别降低19％（P〈0．05）、52％（P〈0．01）和37％（P〈0．01），而其表型标志蛋白仅-SMA、Calponin-1表达无明显变化。结论miR-563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。
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 reac- tion （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 pheno- type protein of α-SMA was down-regulated by 28% （P 〈 0. 05 ） ; the expression of miR-363 was down-reg- ulated by 70% （P 〈0. 01 ）. Compared to miR-NC group, significant inhibition of proliferation and migra- tion were observed in miR-363 over-expressed VSMCs, by 32% and 30% , respectively at 72 hours （P 〈 O. 05）. Krnppel-like factor 4 （ Klf4）, the target gene of miR-363, was down-regulated by 19% （P 〈 O. 05） , 52% （ P 〈 0. O1 ） and 37% （ P 〈 O. 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. Con- clusion 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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第4期702-705,共4页
Chinese Journal of Experimental Surgery
