Fig. 5

LINC02609 /miR-149-5p regulates Apol1 expression in ccRCC cells. (A) Subcellular distribution of LINC02609 in 786-O and A498 cells. GAPDH was used as cytoplasm control and U6 was used as nucleus control. (B) RNA fluorescence in situ hybridization (FISH) showed that LINC02609 was predominantly localized in cytoplasm. U6 was mainly localized in nucleus, used as negative control. 18 S was mainly localized in cytoplasm, used as positive control. LINC02609, U6, and 18 S probes were labeled with Cy3, Nuclei was stained with DAPI. (C) Schematic miR-149-5p putative target sites in 3′ UTRs of LINC02609 and APOL1. (D) Luciferase reporters harboring putative target sites in the 3′ UTRs of APOL1 were co-transfected with 50 and 100 nM of miR-149-5p mimics or miR-149-5p mutant mimics in 786-O and A498 cells. (E) Luciferase reporters harboring putative target sites in the 3′ UTRs of LINC02609 were co-transfected with 50 and 100 nM of miR-149-5p mimics or miR-149-5p mutant mimics in 786-O and A498 cells. Relative luciferase activity was plotted as the mean ± SEM of three independent experiments. (F, G) Q-RT-PCR analysis of APOL1 and LINC02609 in renal cancer cell 786-O and A498 after transfected with miR-149-5p mimics. (H) Western blotting analysis of APOL1 expression in indicated cells. GAPDH was used as a loading control. (I) Q-RT-PCR analysis of APOL1 in renal cancer cell 786-O and A498 with LINC02609 knockdown. (J) WB analysis of the protein levels of APOL1 in response to deregulated LINC02609 expression of indicated cells