miR-103 and miR-107 are involved in the regulation of CDK5R1/p35 expression

CDK5R1 encodes for p35, an activator of CDK5, which is involved in neuronal migration and differentiation during CNS development. A lot of microRNAs (miRNAs) target sites for twenty different miRNAs have been predicted by PicTar in CDK5R1 3’UTR. A Real-Time PCR of 5 selected miRNAs performed in human cell lines showed an inverse correlation between miR-103 and miR-107 levels and p35 expression, suggesting a negative effect of these miRNAs on CDK5R1 expression. We carried out transient transfection of SK-N-BE neuroblastoma cells with miR-107/103 precursors (pre-miR-107/103) and antagonists (anti-miR-107/103). 48h after pre-miR-107 transfection, a significant reduction of p35 levels was observed, by 38% with 50nM and 49% with 100nM of precursor. Otherwise, if anti-miR-107 (50-100nM) was transfected, an increase of 1.4-1.8 folds in p35 levels was observed, compared to the untransfected control. Similarly, the transfection of pre-miR-103 (50-100nM) or anti-miR-103 (50-100nM) caused a reduction (35-40%) or an increase (76-83%) of p35 levels, respectively. Luciferase assays on 3’UTR constructs cotransfected with miR-103/107 showed that 4 out of 9 predicted target sites might be functional. Deletion of the binding sites is in progress: at present deletion of site 1 shows increasing of luciferase activity, while deletion of site 2 produces no effects. Finally, transfection of miR-103 or miR-107 reduces SK-N-BE migration ability by 47 and 45% respectively following in vitro scratch assays. The obtained findings indicate that miR-103 and miR- 107 regulate CDK5R1/p35 expression and allow us to hypothesize that a miRNA-mediated mechanism might influence CDK5 activity and its pathway