采用双荧光素酶报告法验证乳腺癌中miR-155靶标  

Identification of MiR-155 Target in Breast Cancer by Dual-Luciferase Reporter Assay

作　　者：孟丽[1] 纪婷[1] 李鹿丰[1] 张喜平[2] 杨红健[2] 朱聪 郭江峰[1] 丁先锋[1] 

机构地区：[1]浙江理工大学生物工程研究所,杭州310018 [2]浙江省肿瘤医院,杭州310022 [3]浙江省微生物研究所,杭州310012

出　　处：《中国生物化学与分子生物学报》2013年第2期189-195,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省自然科学基金(No.Y2100681);杭州市科技发展计划(No.20110733Q04;No.20110733Q21);公益技术研究社会发展项目(No.2012C23072)~~

摘　　要：MiR-155与乳腺癌发生发展密切相关.本研究以乳腺癌组织和血清中均显著高表达的miR-155为研究对象,利用TargetScan和PicTar预测miR-155的靶基因.根据miR-155与靶基因结合的保守性、动力学及靶基因功能,筛选出miR-155的靶基因ACTA1(actin alpha 1,skeletal muscle)和CEBPB(CCAAT/enhancer binding protein beta),并用双荧光素酶报告法验证.将ACTA1和CEBPB的3'-UTR(3'-untranslated region)全长序列载入海肾荧光素酶基因的下游,并构建结合位点的突变序列,得到pRL-TK-Aw、pRL-TK-Am、pRL-TK-Cw、pRL-TK-Cm载体.不同海肾荧光素酶载体转染Bcap37乳腺癌细胞,同时转染miR-155及内参对照萤火虫荧光素酶载体pGL3-control.根据不同转染的海肾荧光素酶表达活性,运用SPSS软件分析,结果显示,CEBPB是miR-155在乳腺癌中的直接靶基因(P<0.05).miR-155通过下调CEBPB影响乳腺癌的发生.MiR-155 was up-regulated in both tissue and serum sample in breast cancer patients, which may induce breast tumorigenesis. ACTA1 （actin, alpha 1, skeletal muscle）and CEBPB （CCAAT/ enhancer binding protein beta）were predicted as the potential target genes of miR-155 by TaregetScan and PicTar, screened based on the complement conservation and dynamics and identified by dual- luciferase reporter assay. Renilla luciferase vectors of 3＇-UTR of ACTA1/CEBPB and their mutations were constructed, and then transferred into breast cancer cells, Beap37. Simultaneously, miR-155 mimics and the control vector pGL3-control were transferred into the cancer cells. The renilla luciferase expression activity which strongly influenced by the reaction the fluorescence intensity. Data was standardized of miR-155 and its targets, was detected by measuring and analyzed by SPSS. Statistical analvsis identified the CEBPB but not ACTA1 is the target of miR-155（P 〈0.05）.

关 键 词：MIR-155 靶基因 双荧光素酶报告法 CEBPB 

分 类 号：Q81[生物学—生物工程] Q7