Let-7a对尤文氏肉瘤细胞生物学行为的影响  

The efects of microRNA let-7a on biological behaviors of Ewing's sarcoma in vitro

作　　者：张中卒[1] 黄路[2] 虞志明[1] 陈翔[1] 张政[3] 韩智敏[1] 杨东[1] 詹平[1] 曹凯[1] 

机构地区：[1]南昌大学第一附属医院骨科,江西南昌330006 [2]江西省妇幼保健院儿童保健科,江西南昌330006 [3]重庆医科大学细胞生物学及遗传学教研室,重庆400016

出　　处：《肿瘤》2013年第11期939-946,共8页Tumor

基　　金：江西省自然科学基金资助项目(编号:2009GQY0204)

摘　　要：目的:探讨微小RNA(microRNA,miRNA)let-7a对尤文氏肉瘤A673和SK-ES-1细胞生物学行为的影响及其可能的作用机制。方法:将成熟的let-7a片段(has-miR-let-7a mimic)转染至A673和SK-ES-1细胞中,实验分为转染组(转染has-miR-let-7a mimic)、对照组(转染has-miR-let-7a mimic-control)和未转染组(仅加入脂质体)。Has-miR-let-7a mimic转染后,应用实时荧光定量-PCR法检测细胞中let-7a的表达,CCK-8法检测各组细胞的增殖活性,FCM法检测细胞的周期分布和凋亡变化,Transwell小室检测细胞的迁移和侵袭能力,蛋白质印迹法检测细胞中细胞周期依赖性激酶6(cyclin dependent kinase 6,CDK6)、Rb和磷酸化-Rb(phospho-Rb,p-Rb)蛋白的表达。结果:与未转染组和对照组比较,转染has-miR-let-7a mimic后的A673和SK-ES-1细胞中let-7a的表达水平明显上调(P<0.01),细胞增殖受到抑制(P<0.01),G0/G1期细胞所占比例上升(P<0.01),细胞的早期凋亡率明显升高(P<0.01),细胞的迁移和侵袭能力明显下降(P<0.01),细胞中CDK6和p-Rb蛋白的表达水平明显下降(P<0.01),而Rb蛋白的表达水平无明显变化。结论:Let-7a可抑制尤文氏肉瘤A673和SK-ES-1细胞的增殖、迁移和侵袭,并促进其凋亡,这一作用可能部分依赖于let-7a靶向抑制CDK6的表达。Objective： To investigate the effects of microRNA let-7a on the biological behaviors of Ewing’s sarcoma cell lines A673 and SK-ES-1, and to explore the possible mechanisms. Methods： Has-miR-let-7a mimic was transfected into A673 and SK-ES-1 cells. Three groups were designed in this study, including let-7a （transfected with has-miR-let-7a mimic）, control （transfected with has-miR-let-7a mimic-control） and the untreated （transfected with liposomes） groups. The expression levels of let-7a in A673 and SK-ES-1 cells after transfection with has-miR-let-7a mimic were examined by real-time fluorescence quantitative-PCR. The proliferation, migration and invasion abilities were detected by cell counting kit （CCK-8） and Transwell chamber assay, respectively. The cell cycle distribution and the apoptotic rates of A673 and SK-ES-1 cells were measured by flow cytometry. The expression levels of cyclin-dependent kinase 6 （CDK6）, Rb and p-Rb proteins in A673 and SK-ES-1 cells were detected by Western blotting. Results： As compared with the control group and the untreated group, the expression levels of let-7a in A673 and SK-ES-1 cells after transfection with has-miR-let-7a mimic were up-regulated （P 〈 0.01）, and the abilities of proliferation, migration and invasion were inhibited （all P 〈 0.01）.The cell cycle was arrested at G0/G1-phase （P 〈 0.01） and the early apoptosis was increased （P 〈 0.01）. The expression levels of CDK6 and p-Rb were down-regulated （P 〈 0.01）, but the expression level of Rb had no change. Conclusion： Let-7a may inhibit the proliferation, migration and invasion abilities of Ewing’s sarcoma A673 and SK-ES-1 cells and promote the apoptosis. This effect may be partially related to let-7a targeting the inhibition of CDK6 expression.

关 键 词：肉瘤 EWING 微RNAS 转染 Let-7a 细胞 A673 细胞 SK-ES-1 

分 类 号：R738.1[医药卫生—肿瘤]