微小RNA-144上调Rb抑制骨肉瘤细胞增殖及诱导凋亡  

miR-144 supresses proliferation and induces apoptosis of osteosarcoma cells by upregulating Rb

作　　者：颜茂华[1] 许斌[1] 赵立来[1] 朱求亮[1] 罗建民[1] 杨正明[2] 

机构地区：[1]浙江省安吉县人民医院骨科,浙江安吉313300 [2]浙江大学医学院附属第二医院,浙江杭州310009

出　　处：《中国现代医生》2015年第6期6-9,12,F0003,共6页China Modern Doctor

基　　金：浙江省医药卫生科技计划项目(2010KYB052)

摘　　要：目的探讨微小RNA-144(miR-144)对骨肉瘤细胞增殖、凋亡及相关蛋白影响。方法运用脂质体转染的方法,在骨肉瘤细胞株MG-63中转染不同浓度miR-144类似物(miR-144组),随机miR-144片段作为阴性对照组。实时定量PCR检测miR-144表达水平。DAPI染色观察miR-144对细胞形态的影响,用MTT检测细胞增殖,Annexin/PI双染确定细胞凋亡比例。免疫印迹检测PTEN以及Rb等抑癌基因编码蛋白和凋亡相关蛋白表达情况。结果实时定量PCR结果显示,miR-144组miR-144的表达(1128.54±738.52)明显高于空白对照组(1.00±0.00)和阴性对照组(2.06±0.73)(P<0.01)。与阴性对照组和空白对照组比较,miR-144组细胞数量明显减少,增殖受抑制,流式细胞检测显示,细胞凋亡率明显增加。免疫印迹检测显示,Rb和PTEN表达水平有所增加,而凋亡相关的Caspase-3活化,线粒体膜上Cyto C释放。而阴性对照组与空白对照组以上数值比较,差异均无统计学意义(P>0.05)。结论合成的miR-144片段(miR-144 mimic)能顺利进入细胞并上调miR-144的表达,对骨肉瘤细胞都有一定程度的抑制作用,并有浓度依赖性,对MG-63的抑制效果最强,并通过活化Caspase-3促进凋亡,上调PTEN和Rb的表达实现对细胞的抑制作用。未来可以基于miR-144进行骨肉瘤治疗的短核苷酸类药物开发。Objective To study the influences of microRNA-144 on osteosarcoma cells from proliferation, apoptosis and the expression of related protein. Methods Different concentration of miR-144 was transfected into osteosarcoma MG-63 cell lines by the method of lipofectmine. The cell lines were divided into 3 groups： normal group, miR-144 transfected group, and negative group which was transfected with random miR-144 fragment. The proliferation of cells was detected by the MTT assay. The expression of miR-144 was detected by Real-Time PCR. DAPI staining was carried to study the influence of miR-144 on the morphology of MG-63. And the percentage of apoptosis was observed by flow cytometry with the Annexin/PI staining. Expression of tumor suppressor such as PTEN and Rb and the protein related with apoptosis were evaluated by western blotting. Results RT-PCR indicated a higher expression of miR-144 in transfected group（1128.54±738.52） compared with the normal group（1.00 ±0.00） and negative group（2.06±0.73）（P〈0.01）. The proliferation of miR-144 transfected cells was significantly lower than that in normal group and negative group. Further, a higher apoptosis rate was detected by flow cytometry with a statistically significant upregulation of PTEN, Rb, Caspase-3 and Cyto C in transfected group, indicating an internal apoptosis pathway was involved in this process. Moreover, no significant changes were found in the normal group and negative group（P〉0.05）. Conclusion miR-144 could transfectd into cells by the method of liposomal transfection and in turn upregulate the expression of miR-144 mimic, with an inhibition on the proliferation of MG-63. The underlying mechanisms may relate to the upregulation of tumor suppressor and activation of protein associated with caspase-3 signaling pathway, with providing a novel horizon in short nucleotide drugs on the management of osteosarcoma.

关 键 词：骨肉瘤 微小RNA-144 增殖 凋亡 

分 类 号：R738.1[医药卫生—肿瘤]