FIGURE 6.

PUF60 promoted bladder cancer cell growth via transcriptionally upregulating AURKA expression. (A) PUF60 and AURKA expression was detected by RT-qPCR and western blot in 5637 cells with PUF60 knocked down (PUF60: NC vs. Si-1: P < 0.0001, NC vs. Si-2: P < 0.0001; AURKA: NC vs. Si-1: P = 0.0005, NC vs. Si-2: P < 0.0032). (B) PUF60 and AURKA expression was detected by RT-qPCR and western blot in T24 cells overexpressing PUF60 (PUF60: OE vs. vector: P < 0.0001; AURKA: OE vs. vector: P = 0.0033). (C) Ideograph of different segments of AURKA promoter. (D) Different segments of AURKA promoter were amplified by PCR using specific primers, and PCR products were detected by agarose gel electrophoresis. (E) Relative promoter activity of different segments of AURKA promoter measured by dual luciferase assay. (F) Relative activity of AURKA promoter was measured after knockdown of PUF60 in 5637 cells (NC vs. Si-1: P < 0.0001, NC vs. Si-2: P < 0.0001). (G) Relative activity of AURKA promoter was measured after overexpression of PUF60 in T24 cells (OE vs. vector: P < 0.0001). (H) Binding of PUF60 on the 5′-biotin labeled AURKA promoter probe or a control non-specific probe was detected by Western blot using anti-PUF60 antibody. (I,J) Knockdown of PUF60 inhibited the clonogenicity (I) and viability (J) of 5637 cells, which was reversed by AURKA overexpression (I: NC vs. sh: P < 0.0001, sh + vector vs. sh + AURKA: P < 0.0001; J: NC vs. sh: P < 0.0001, sh + vector vs. sh + AURKA: P < 0.0001). (K,L) Overexpression of PUF60 promoted the clonogenicity (K) and viability (L) of T24 cells, which was reversed by AURKA inhibitor (K: OE vs. vector: P < 0.0001, OE + DMSO vs. OE + AURKAi: P < 0.0001; L: OE vs. vector: P < 0.0001, OE + DMSO vs. OE + AURKAi: P < 0.0001). Clonogenicity was determined by colony formation assay. Viability was measured by MTS assay. Data was analyzed by t-test; **P < 0.01, ***P < 0.001, ****P < 0.0001.