microRNA-136影响静脉平滑肌细胞增殖和迁移的作用机制  

MircroRNA-136 suppresses proliferation and migration of venous smooth muscle cells by targeting Steap3

作　　者：杨立国[1] 刘萃萃[2] 谭娟娟[3] 马江伟[4] 张立[4] 葛广豪[4] 刘化进[4] 严志强[2] 乔增勇[4] 

机构地区：[1]南方医科大学第三临床学院,广东广州510515 [2]南方医科大学附属奉贤医院中心实验室 [3]上海交通大学生命科学技术学院 [4]南方医科大学附属奉贤医院心内科

出　　处：《上海医学》2016年第1期29-34,I0002,共7页Shanghai Medical Journal

基　　金：国家自然科学基金资助项目(11172176)

摘　　要：目的探讨microRNA-136(miR-136)对静脉平滑肌细胞增殖、迁移的影响及其作用机制。方法建立大鼠颈总动脉自体移植静脉模型,并于体外培养静脉平滑肌细胞。采用CCK(cell counting kit)-8和EDU(5-Ethynyl-2’-deoxyuridine)实验检测细胞增殖情况,Transwell和细胞划痕实验检测细胞迁移情况,荧光定量PCR检测移植1、2、4周后和体外培养静脉平滑肌细胞中miR-136的相对表达量,免疫印迹法检测Steap3蛋白质表达量,免疫荧光法检测蛋白Steap3在细胞内的定位情况。通过microRNAs专业网站预测筛选可能的下游基因,并利用双荧光素酶报告基因验证。结果荧光定量PCR结果显示,移植1、2周后移植静脉miR-136的相对表达量分别为0.46±0.33、0.37±0.30,显著低于对侧未处理静脉(P值分别<0.05、0.01);移植4周后移植静脉miR-136的相对表达量为1.39±0.82,与对侧未处理静脉的差异无统计学意义(P>0.05);处于增殖状态的静脉平滑肌细胞中miR-136的相对表达量为0.11±0.01,显著低于处于非增殖状态的细胞(P<0.05)。CCK-8实验结果显示,阴性对照(NC)+血清组的光密度(D)值为0.96±0.03,显著高于NC组的0.62±0.02和模拟物+血清组的0.76±0.08(P值均<0.05)。EDU实验结果显示,NC+血清组EDU标记的阳性细胞百分比为(52.71±2.57)%,显著高于NC组的(13.91±3.43)%和模拟物+血清组的(32.56±3.26)%(P值均<0.01)。Transwell实验结果示,NC+血清组细胞迁移数为(53.13±16.36)个/视野,显著多于NC组的(8.09±5.44)个/视野和模拟物+血清组的(8.08±0.85)个/视野(P值均<0.05);细胞划痕实验结果显示,NC+血清组细胞24h迁移率为(45.40±1.17)%,显著高于NC组的(19.27±0.31)%和模拟物+血清组的(22.36±5.55)%(P值均<0.01)。免疫印迹法检测结果显示,NC+血清组的Steap3蛋白质相对表达量为0.91±0.07,显著高于NC组的0.52±0.10和模拟物+血清组的0.15±0.01(P值均<0.01)。免疫荧光染色结果显示,Steap3蛋白质主要定位于细胞�Objective To investigate the potential role and mechanism of microRNA-136（miR-136）in suppressing the proliferation and migration of venous smooth muscle cells（VSMCs）.Methods Autogenous common carotid artery vein graft model were established in rats and VSMCs were cultured in vitro.Cell counting kit-8（CCK-8）and 5-Ethynyl-2＇-deoxyuridine（EDU）assay were used to detect cell proliferation.Transwell and cell wound Healing assay were used to determine cell migration.The miR-136 expression was detected using realtime polymerase chain reaction（PCR）.Steap3 protein expression was measured by Western blotting.The location of Steap3 protein was determined by immunofluorescence.Downstream targeted genes were predicted and screened in bioinformatics website of microRNAs;and were verified by dual-luciferase reporter gene assay.Results The miR-136 expression of grafted vein was 0.46±0.33,0.37±0.30 and 1.39±0.82 on week 1,2 and 4 after transplantation,respectively.As compared with contralateral ungrafted veins,the expression of miR-136 was significantly decreased in grafted veins on week 1and 2 after transplantation（P〈0.05,0.01）,while there was no significantly difference on week 4（P〉0.05）.In addition,the miR-136 expression was 0.11±0.01 in proliferative VSMCs,which was significantly lower than that in non-proliferative VSMCs（P〈0.05）.CCK-8assay showed that the optical density values（D450）in NC＋serum group was 0.96±0.03,which was significantly higher than that in NC group（0.62±0.02）and Mimic＋serum group（0.76±0.08,both P〈0.05）.The percentage of positive cells labelled by EDU in NC＋serum group was（52.71±2.57）%,which was significantly higher than that in NC group（13.91±3.43）% and Mimic＋serum group（／[32.56±3.26／]%,both P〈0.01）.Transwell assay showed that the number of migrated cells in NC＋serum group was 53.13±16.36 per field,which was significantly more than that in NC group（8.09±5.44 per field）and Mimic＋serum group（8.08±0.85 per fie

关 键 词：microRNA-136 新生内膜增生 静脉平滑肌细胞 增殖 迁移 

分 类 号：R654[医药卫生—外科学]