Determination of mRNA Targets of miR-376c within the RNA-Induced Silencing Complex

The focus of the present study was to establish an easy combined method based on the MirTrap system together with a mRNA array to efficiently define target mRNAs which can be bound by miR-376 within the RNA-induced silencing complex (RISC) as well as to deduce the biological relevance of this miRNA in modulating cancer-associated signaling pathways. The importance of this study is to analyze miR-376/mRNA pairs which are imbedded in RISC, thus in their natural environment using the MirTrap system. The subsequent application of a mRNA array, instead to use the elaborate next generation sequencing approach, eases the identification of mRNA targets that can be bound by a miRNA within RISC. MRNA target screening was carried out by the MirTrap system. The breast cancer cell line MDA-MB-468 was transfected with plasmids encoding for miR-376c and RISC. MiR-376c bound to mRNAs and integrated in RISC were immuno-precipitated with an antibody against RISC and identified by a mRNA array containing 89 different mRNAs. Western blot were performed to verify the repression of these target mRNAs by miR-376c. We found 56 potential mRNA targets for miR-376c within RISC. Particularly strong binding activities of miR-376c were to the matrix metalloproteinase MMP9, the apoptosis inhibitor BCL2 and the cell cycle inhibitor CDKN1A/p21. Western blotting showed a slight and strong repression of protein expression of BCL2 and p21, respectively. The different strength of protein repression is possibly owing to the overexpression of RISC. Our findings show a variety of potential target mRNAs for miR-376c within RISC with different binding activities. MiR-376c is able to bind tumor suppressive and oncogenic mRNAs.