FIGURE 2.

m⁶A regulates mRNA stability and translation of ZNF677 in renal cell carcinoma (RCC) cells. (A and B) Western blot (A) and RT‐qPCR (B) analysis of ZNF677 expression in different RCC cell lines (786‐O, OSRC, 769‐P, CAKI1, CAKI2) compared to normal epithelium cells of renal tubule HK2 cell. (C) MeRIP‐qPCR analysis of ZNF677 m⁶A levels in HK2, OSRC and CAKI2 cells. (D and E) OSRC and CAKI2 cells were transfected with vector control or Mettl3 construct for 24 h, the protein and mRNA expression levels of ZNF677 were measured by Western blot (D) and RT‐qPCR (E), respectively. (F and G) MeRIP‐qPCR analysis of ZNF677 m⁶A levels in control and overexpression of Mettl3 OSRC (F) and CAKI2 cells (G). (H) After treatment with Act‐D for the indicated times, the mRNA levels of ZNF677 were checked in control and Mettl3‐overexpressed OSRC cells. (I) OSRC cells were pretransfected with vector control or Mettl3 construct for 24 h and then further treated with CHX (10 μg/ml) or MG‐132 (5 μM) for 6 h, the expression of ZNF677 was detected by Western blot analysis. (J and K) Firefly (F‐Luc) values were normalised against Renilla luciferase levels, and ZNF677 translation efficiency was calculated for the pmirGLO‐ZNF677 reporter relative to pmirGLO in Mettl3 overexpression and control OSRC cells. NS, not significant; *p < .05 or **p < .01 indicates a significant difference between the indicated groups