采用双荧光素酶报告法验证乳腺癌中miR-155靶标

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.

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

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, Bcap37. 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 of miR-155 and its targets, was detected by measuring the fluorescence intensity. Data was standardized and analyzed by SPSS. Statistical analysis identified the CEBPB but not ACTA1 is the target of miR 155(P<0.05).