Fig. 8

CircMET modulates SMAD3 expression through post‑transcriptional regulation. (A) Schematic of the selection for the direct downstream target of miR-1197. (B) HEK293T cells were co-transfected with 3’-UTR luciferase truncations of top50 mRNA and miR-1197, and the luciferase activity was determined using a dual luciferase reporter assay after 48 h. (C) RIP and MS2-RIP assays were performed using AGO2/GFP antibody in UOK109 cells, then the enrichment of SMAD3 mRNA was detected by real-time PCR. (E) The miR-1197-Biotin complex was enriched by Biotin-antibody with protein extracts from UOK109 and 786-O cells. The enrichment of SMAD3 and miR-1197 was detected by real-time PCR. (F) Schematic illustration of SMAD3 3’-UTR wild type and mutation luciferase reporter vectors. (G) HEK293T cells were co-transfected with miRNA mimics respectively and wild-type or mutant SMAD3 3’-UTR luciferase reporter vector, and luciferase reporter activity was detected. (H–O) The RNA levels of miR-1197 and SMAD3 and the protein level of SMAD3 were detected after transfection with indicated lentivirus. (P) Cell viability of UOK109 and 786-O cells was determined using CCK-8 assays after transfection for 48 h. (Q-R) A colony formation and tumor sphere formation assay were used to determine the colony and tumor sphere formation ability of UOK109 and 786-O cells co-transfected with indicated lentivirus. (S) Cell cycle analysis was performed using flow cytometry in cells transfected with indicated lentivirus. The data are presented as the mean ± SD, n.s. = non-significant, *P < 0.05, ***P < 0.001