Fig. 5.

CircMET down‑regulates CDKN2A mRNA by direct binding. (A) HEK293T cells were co-transfected with indicated mRNA 3’-UTR luciferase truncations and sh-circMET, and the luciferase activity was determined using a dual luciferase reporter assay after 48 h. (B) Dual luciferase assay of HEK293T cells co-transfected with firefly luciferase constructs containing the wild-type or mutant AAUAAA motif in CDKN2A mRNA 3’-UTR and circMET plasma were performed. (C) Dual luciferase assay of HEK293T cells co-transfected with CDKN2A mRNA 3’-UTR luciferase constructs and wild-type or mutant circMET plasma were performed. (D) The RNA levels of circMET and CDKN2A mRNA in both UOK109 cells and 786-O cells were detected by real-time PCR after silence or overexpression of circMET. (E) RNA pulldown and MS2-RIP assays were performed to confirm the association of CDKN2A mRNA with circMET. (F) The RNA levels of circMET and CDKN2A mRNA in both UOK109 cells and 786-O cells were detected by real-time PCR after silence or overexpression of NONO-TFE3. (G-L) The stability of CDKN2A mRNA in cells transfected with indicated lentivirus after treatment with α-amanitin. (M) The protein level of CDKN2A in both UOK109 cells and 786-O cells were detected by western blot after silence or overexpression of circMET. The data are presented as the mean ± SD, n.s. = non-significant, **P < 0.01, ***P < 0.001