Fig. 6.

circAGAP1 regulated the miR-15a-5p/E2F3 pathway in ccRCC. a The expression levels of E2F3 in paired ccRCC and adjacent noncancerous tissues were analyzed with samples from the TCGA database (p = 0.0002). b The relationship between the levels of circAGAP1 and overall survival was analyzed by Kaplan-Meier survival analysis. C mRNA levels of E2F3 in ccRCC and adjacent noncancerous tissues detected by qRT-PCR (n = 34, p = 0.0149). d, e Negative associations were found between miR-15a-5p and E2F3 expression in ccRCC, whereas a positive association was found between circAGAP1 and E2F3 expression in ccRCC. f The effects of circAGAP1 overexpression (OE) or knockdown (siRNA) on E2F3 protein levels in A498 and ACHN cells detected by Western blotting. g, h WT and MUT sequences of the potential binding sites of miR-15a-5p in E2F3. The impact of miR-15a-5p mimics on the luciferase activities of WT-E2F3 and MUT-E2F3 in cells was detected. i Influence of circAGAP1 and miR-15a-5p on the protein levels of E2F3, N-cadherin, PCNA, and Bcl-2 in A498 and ACHN cells as detected by WB. j Influence of different hypoxia treatments on E2F3 protein levels in A498 and ACHN cells as detected by WB. Cells were subjected to either normoxia or hypoxia for 12 h, 24 h and 48 h. k Overexpression of E2F3 in ACHN and A498 cells as detected by WB. l The viability of A498 and ACHN cells transfected with mock or E2F3 as detected by CCK-8. m EdU analysis and quantification of EdU-positive A498 and ACHN cells transfected with mock or E2F3. Cells were subjected to hypoxia for 24 h. n Apoptosis rates of A498 and ACHN cells transfected with mock or E2F3 by flow cytometry. o, p Transwell analyses of A498 and ACHN cells transfected with mock or E2F3. The experiments were repeated with at least three biological replicates. **p < 0.01