南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (03): 368-.

• • 上一篇    下一篇

miRNAs慢病毒文库筛选靶向EZH2 3’非翻译区的新型miRNAs及其在乳腺癌中的表达

刘萃萃，王璐璐，赵卫卫，彭攸，王玉平，孙振亮，冯景   

  • 出版日期:2014-03-20 发布日期:2014-03-20

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

  • Online:2014-03-20 Published:2014-03-20

PDF

1796

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。

Abstract: Objective To screen novel miRNAs targeting EZH2 3’ untranslated region (UTR) in recombinational MCF-7 breast
cancer cells over-expressing EZH2 3’ 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<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’ UTR. These miRNAs are expressed differentially between normal breast
cells and breast cancer tissues.