Figure 4

Candidate p53 network genes that were positive in both the GOF/LOF screen and miR-125b pull-down were validated for targeting by miR-125b using the 3′ UTR luciferase reporter assay and Western blots for protein expression. (A-C), Reporter genes containing the full-length 3′ UTRs of each selected target gene were co-transfected with miR-125b duplex into 293T cells. Luciferase readings were obtained 48 hours after transfection and presented here as the average percentage of luciferase activity ± s.e.m. (n≥3) relative to a scrambled duplex co-transfected control (100%). A reporter containing a 23-nucleotide-binding-site with perfect complementarity to miR-125b was used as the perfect match positive control, while the unmodified luciferase reporter was used as the empty negative control. (A) Human: 10 out of 13 candidate genes' 3′ UTRs showed significant repression by miR-125b relative to the control (p<0.01). (B) Mouse: 9 out of 11 candidate genes' 3′ UTRs showed significant repression by miR-125b relative to the control (p<0.01). (C) Zebrafish: 7 out of 8 candidate genes' 3′ UTRs showed significant repression by miR-125b relative to the control (p<0.01). (D) Alignment of predicted miR-125b binding sites in the 3′UTRs of Ppp1ca, Prkra and Tp53 across three species. Seed-binding sequences are underlined. Bases conserved in two (blue) or three (black) species are highlighted. (E) The 3′UTR seed-binding sequences of 7 target mRNAs were mutated and assayed for direct binding to miR-125b using the luciferase reporter assay, relative to wild-type 3′UTR sequences. (E) The seed-binding sequences in the 3′UTR of 7 predicted target mRNAs were mutated and compared to wild-type sequences for binding to miR-125b using luciferase reporter assay. (F-G) Western blot analysis of protein expression of selected target genes two days after a transfection of miR-125b duplex, miR-125b antisense (AS) or negative control duplex or negative control antisense. (F) Western blots showed that miR-125b repressed BAK1, PPP1CA, TP53, and PPP2CA levels in human SH-SY5Y neuroblastoma cells, while the antisense RNA miR-125b-AS derepressed expression of these proteins in human ReNcell VM neural progenitor cells. (G) Western blots showed that miR-125b repressed BAK1, PPP1CA, PUMA, and ITCH levels in mouse N2A neuroblastoma cells. Abbreviations: h, human; m, mouse; z, zebrafish.