FIGURE 6.

ZNF677 transcriptionally regulates expression of CDKN3. (A) The mRNA expression of potential target genes of ZNF677 in OSRC cells transfected with dCas13b‐METTL3 combined with gRNA control or gRNA for ZNF677 for 24 h were checked by RT‐qPCR analysis. (B) The mRNA expression of potential target genes of ZNF677 in OSRC cells transfected with siRNA control or siRNA for ZNF677 for 24 h were checked by RT‐qPCR analysis. (C) The protein expressions of ZNF677 and CDKN3 in OSRC and CAKI2 cells transfected with dCas13b‐METTL3 combined with gRNA control or gRNA for ZNF677 for 24 h were checked by Western blot analysis. (D) The protein expressions of ZNF677 and CDKN3 in OSRC and CAKI2 cells transfected with siRNA control or siRNA for ZNF677 for 24 h were checked by Western blot analysis. (E) The binding between ZNF677 and promoter of CDKN3 was checked by ChIP‐qPCR using IgG or ZNF677 antibody. (F) Schematic representation of the potential binding sites 1, 2 and 3 between ZNF677 and the promoter of CDKN3. (G) Binding between ZNF677 and the promoter of CDKN3 at the potential binding sites 1, 2 and 3 in OSRC cells transfected with siRNA control or siRNA for ZNF677 for 24 h was checked by ChIP‐qPCR. (H) Schematic representation of the mutated CDKN3 promoter reporter to investigate the role of ZNF677 in CDKN3 expression. (I) OSRC cells were co‐transfected with CDKN3‐promoter‐WT, CDKN3‐promoter‐MUT1, CDKN3‐promoter‐MUT2 and si‐NC or si‐ZNF677 for 24 h. Results were presented as the ratio between the activity of the reporter plasmid. (J) Correlation between ZNF677 and CDKN3 in renal cell carcinoma (RCC) patients from ChIPBase database. Data are presented as the mean ± SD from three independent experiments. NS, not significant; *p < .05 or **p < .01 indicates a significant difference between the indicated groups