miRNAs慢病毒文库筛选靶向EZH23'非翻译区的新型miRNAs及其在乳腺癌中的表达  

Screening of novel miRNAs targeting EZH2 3' untranslated region using lenti-miR viruslibrary and their expressions in breast cancer cells and tissues

作　　者：刘萃萃[1,2] 王璐璐[2] 赵卫卫[2] 彭攸[2] 王玉平[2] 孙振亮[2] 冯景[2] 

机构地区：[1]南方医科大学第三临床医学院,广东广州510515 [2]南方医科大学附属奉贤医院检验科,上海201400

出　　处：《南方医科大学学报》2014年第3期368-372,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81202104);上海市自然科学科学基金面上项目(12ZR1426300);上海市卫生系统优秀学科带头人培养计划(XBR20130114)~~

摘　　要：目的重组过表达EZH2基因3'非翻译区的MCF-7细胞株中筛选靶向miRNAs,并进行细胞和组织定量分析。方法重组细胞株感染慢病毒文库,利用细胞毒性药物筛选后抽提细胞基因组,以此为模板PCR扩增出miRNA前体,测序确定miRNAs名称,并对其进行PCR定量分析。结果在重组细胞株中共筛选出7种miRNAs,依次为miR-15b、miR-16-2、miR-181b2、miR-217、miR-224、miR-329-1、miR-487b,这些新型miRNAs用生物信息学软件PicTar和TargetScan无法预测。Real-time PCR发现,与乳腺癌细胞MCF-7相比,正常乳腺细胞株HBL-100中miR-217、miR-329-1、miR-487b高表达;乳腺癌组织中仅miR-15b及miR-16-2表达增加,与癌旁组织相比差异有统计学意义(P<0.05)。结论在重组过表达EZH2 3'-UTR的乳腺癌MCF-7细胞株中结合慢病毒文库可筛选出用生物信息学软件未预测的新型靶向miRNAs,这些新型miRNA在正常乳腺细胞与肿瘤细胞及组织中存在差异表达。Objective To screen novel miRNAs targeting EZH2 3＆#39; untranslated region （UTR） in recombinational MCF-7 breast cancer cells over-expressing EZH2 3＆#39; UTR and quantitative analyze the expressions of the screened miRNA in breast cancer cells and tissues. Methods A lentiviral library was transfected into the recombinant cell line MCF-7. The cells were screened with cytotoxic agents before extraction of the genome for amplification of the miRNA precursors using PCR. The screened miRNAs were identified with sequence analysis and their expressions were analyzed quantitatively with real-time PCR in breast cancer cells and tissues. Results Seven miRNAs were screened from the recombinant MCF-7 cells, namely miR-15b, miR-16-2, miR-181b2, miR-217, miR-224, miR-329-1, and miR-487b, all of which failed to be predicted by bioinformatics software. Real-time PCR showed that miR-217, miR-329-1, and miR-487b were over-expressed in MCF-7 cells, and the expression of miR-15b and miR-16-2 was significantly increased in cancer tissues compared with the adjacent tissues （P＆lt;0.05）. Conclusions Novel targeted miRNAs that can not be predicted by bioinformatics software were successfully screened from MCF-7 breast cancer cells over-expressing EZH2 3＆#39; UTR. These miRNAs are expressed differentially between normal breast cells and breast cancer tissues.

关 键 词：乳腺癌 EZH2 慢病毒文库 EZH2 

分 类 号：R737.9[医药卫生—肿瘤]