miR-101对骨肉瘤MG-63细胞增殖和凋亡影响机制探讨  

Role and mechanism of miR-101 in proliferation and apoptosis of osteosarcoma cell line MG-63

作　　者：林松[1] 邵楠楠[2] 范磊[1] 马秀才[1] 浦飞飞[1] 刘勇[1] 邵增务[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,湖北武汉430022 [2]郑州大学附属河南省肿瘤医院放射科,河南郑州450008

出　　处：《中华肿瘤防治杂志》2014年第6期428-432,共5页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的:通过调控微小RNA-101(miR-101)在骨肉瘤细胞MG-63中的表达,探讨miR-101对MG-63细胞增殖和凋亡的作用及其可能机制。方法:采用实时荧光定量PCR(qRT-PCR)检测miR-101在人骨肉瘤MG63细胞和成骨细胞中的相对表达;化学合成miR-101模拟序列(Mimics)和其阴性对照序列,以脂质体为载体,分别转染骨肉瘤MG63细胞,并设立空白对照组;qRT-PCR检测3组细胞miR-101的表达;MTT法检测3组细胞的增殖;流式细胞仪检测3组细胞凋亡及细胞周期变化;免疫印迹法检测转染后3组细胞自噬相关蛋白Beclin1和LC3B表达。结果:qRT-PCR检测结果显示,成骨细胞miR-101的相对表达(2.56±0.27)是未转染骨肉瘤细胞(1.52±0.20)的1.68倍,P=0.003;转染后qRT-PCR检测结果显示,模拟物转染组miR-101的表达(3.86±0.28)较空白对照组(1.31±0.32)和阴性对照组(1.35±0.31)分别上调了2.95倍和2.86倍,F=70.28,P<0.001。MTT结果显示,miR-101模拟物转染组骨肉瘤细胞增殖速率较空白和阴性对照组明显抑制,P<0.01。流式细胞分析结果显示,模拟物转染组G0/G1期细胞百分比为(13.02±1.05)%,较空白对照组(3.15±0.98)%和阴性对照组(3.26±0.94)%明显增加,P<0.001;模拟物转染组细胞凋亡率为(11.28±1.45)%,较空白对照组(3.02±0.96)%和阴性对照组(3.16±0.78)%明显增加,P<0.001。蛋白印迹结果显示,模拟物转染组自噬相关蛋白Beclin1和LC3B的表达较空白对照组均下降。结论:miR-101在骨肉瘤MG-63细胞中呈低表达,可能与骨肉瘤的发生和发展相关。miR-101可抑制骨肉瘤MG-63细胞的增殖和自噬,诱导细胞凋亡和周期阻滞,其机制可能是通过抑制其自噬而发挥作用。OBJECTIVE：To investigate the role and mechanism of miR-101 in proliferation and apoptosis of osteosar- coma cells by regulating the expression of miR-101 in osteosarcoma cell line MG-63. METHODS.. The expression level of miR-101 in MG63 cells and osteoblasts were detected by qRT-PCR. Synthetic miR-101 sequence （mimics） or negative con- trol sequence （NC） was transiently transfected into MG63 cells with lipofectamine 2000 vector. Meanwhile, non-transfect- ed MG-63 cells acted as a blank control, The expression of miR-101 mRNA in three groups was detected by qRT-PCR af- ter transfection. The effects of miR-101 on MG-63 cell proliferation,apoptosis and cell cycle were evaluated by MTT assay and flow cytometry,respectively. The expression of Beclinl and LC3B protein were analyzed by Western-blot. RESULTS.. qRT-PCR results revealed that the expression of miR-101 in osteoblasts （2. 56±0.27） was 1.68 times of that in MG63 cells （1. 52±0.20,P=0. 003） and miR-101 in the mimics group （3. 86±0.28） was increased by 2.95 fold and 2.86 time scompared with the blank group （1.31±0.32） and the negative control group （1.35±0.31） ,respectively （F=70.28,P〈0. 001）. The MTT assay suggested that the rate of cell proliferation of MG-63 was declined significantly in the mimics group as compared with that in the blank group and the NC group （P〈0.01）. Cell cycle analysis showed that transfection of miR-101 mimics led to a lower proportion of cells in S phase and a higher proportion （13.02±0.14）% in G0/G1 phase compared with the blank groups （3.15±0.98）% and the NC groups （3.26±0.94）% ,indicating that growth of MG63 cells was inhibited by miR-101 due to G0,/G1 cell cycle arrest （P〈0. 001）. In addition, the percentage of apoptosis cells was increased significantly （11.28±1.45） % in miR 101 mimics group than that in the blank group （3.02±0.96） % and the NC group （3.16±0.78）% （P〈0. 001）. Western blot analysis suggested that the miR-101 expression showed an

关 键 词：骨肉瘤 miR-101 转染 细胞增殖 细胞凋亡 

分 类 号：R738.1[医药卫生—肿瘤]