(A) (Left panel) Luciferase assay with HEK 293T cells transfected with a siHuR or a scramble siRNA (siCtr) shows that HuR is involved in the noncanonical regulation mediated by miR-200a on the c-Jun 3′UTR WT. The mean value of the corresponding empty vector was set to one. (Right panel) Western blotting evaluation of siRNA knockdown. Proteins were isolated after wound healing assay and actin serves as loading control. (B) qRT-PCR of c-Jun mRNA on cells transfected with the microRNAs overexpressing plasmids (miR-200a, miR-200b, empty vector) and a siRNA control (siCtr) or a siHuR. In the cells transfected with the siHuR, we completely lost the miR-200a inductive effect on c-Jun mRNA, suggesting an involvement of the RNA-binding HuR in the noncanonical action mediated by miR-200a. The mean value of the corresponding empty vector was set to one. (C) JUN protein level measured by Western blotting. The graphic on the left represents the densitometry analysis of three independent experiments; on the right there is only one representative WB experiment; the empty vector was set to one. (D) qRT-PCR with samples from RNA-immunoprecipitation assay (IP, IgG, OB) show a three times enrichment of HuR/c-Jun mRNA interaction in the presence of miR-200a. The mean value of the corresponding IgG was set to one; OB (only beads) was the negative control. (E) qRT-PCRs on input samples from the RIP assay in HEK 293T cells confirmed that c-Jun mRNA was more abundant in the cells transfected with the miR-200a overexpression plasmid compared to the cells transfected with the empty vector. The mean value of the corresponding empty vector was set to one. (F) Schematic of the Luciferase construct with c-Jun 3′UTR MUT-INV that contains a reciprocal exchange in the sequence bounded by the seed region of miR-200a and miR-200b. The mutated nucleotides are underlined. (G) HEK 293T transfection with reporter construct. c-Jun 3′UTR MUT-INV showed that in the new sequence rearrangement, miR-200b induced the RLuc whereas the miR-200a repressed it. The mean value of the corresponding empty vector was set to one. (H) Luciferase assay performed with HEK 293T cells transfected with a HuR or a scramble siRNA revealed that also with the 3′UTR MUT-INV there was an involvement of HuR in the noncanonical effect. The mean value of the corresponding empty vector was set to one. Data represented the mean ± SD and asterisks (*) (in panels A–E,G,H) indicate statistically significant modulations with respect to empty vector according to paired Student's test. (*) P < 0.05; (**) P < 0.01.
