Identification Of MiR-155Target In Breast Cancer By Dual-Luciferase Reporter Assay

Background, objectives Breast cancer is one of the most common malignancies in women,latest data show that breast cancer is still with the highest incidence, accounting for29%of allnew cases of female cancers, the recurrence and mortality rates are high. So the prevention andtreatment of breast cancer have a crucial effect on female life. Studies show that miRNAmutation, deletion and abnormal expression are closely related to the occurrence anddevelopment of many diseases. MiR-155is positively related with development of breast cancer,however, the mechanism that how miR-155regulates its targets and affects breast cancer is stillnot clear. Therefore, identification of miR-155targets is essential for studying the role ofmiR-155in physiological or pathological processes in breast cancer.Methods Based on our previous studies, miR-155, which is highly expressed in both tissueand serum of breast cancer, is selected in breast cancer for further study. The targets of miR-155,predicted by TargetScan and PicTar were screened from following steps:(1) Choosing the sameones among the predicted targets;(2) According to the different evaluation parameters in twosoftwares,16targets which have high conservatism, dynamics were selected;(3) Using GO,BioGPS, KEGG, HPRD and Target miRNA, combining with mRNA characteristics, expressionsites and functions to select closely related targets, ACTA1and CEBPB. The3’UTR full-lengthsequences of ACTA1, CEBPB and their mutations were inserted into the downstream of theRenilla luciferase gene in pRL-TK vectors, constructed4recombinant vectors: pRL-TK-AW,PRL-TK-Am, pRL-TK-Cw, pRL-TK-Cm. Then4recombinant plasmids were transfected intoBcap37breast cancer cells, miR-155and the control, pGL3-control were transfectedsimultaneously. Based on the Renilla luciferase activities in different transfected cells, miR-155targets were confirmed.Results Renilla luciferase activity analysis using SPSS software indicted that CEBPB butnot ACTA1is the direct target gene of miR-155(P<0.05) in the breast cancer.Conclutions: The down-regulated expression of CEBPB by miR-155affects proliferationindex of breast cancer cell.