FIGURE 7.

CircGNG7 expression level was regulated by SMAD4 binding on GNG7 promoter. (A) The potential transcription factors binding to GNG7 promoter were predicted by using public database LASAGNA‐Search 2.0 with the threshold P value <0.001. (B) The circGNG7 expression was detected by RT‐PCR in CAL27 and HN6 cells transfected with siRNA of selected genes (NF1, LEF1, SMAD3, SMAD4, SPL1, SP1, JUN, NFATC2, TEAD2, E2F1, CREB1, ATF2, ZEB1, WT1, PLAU, NFKB1, and MYB) for 24 h. (C‐D) CAL27 and HN6 cells were transfected with SMAD4 overexpression plasmid (ov‐SMAD4) or vector for 48 h. (C) The circGNG7 expression was evaluated by RT‐PCR. (D) The binding of SMAD4 at the GNG7 promoter region was detected by a chromatin immunoprecipitation assay. (E) CAL27 and HN6 cells were co‐transfected with circGNG7 mutant/wildtype plasmid and ov‐SMAD4, and the luciferase activity was determined by using a dual luciferase reporter assay after 24 h. (F) The expression levels of SMAD4, total HSP27, HSP27‐S15, HSP27‐S78, and HSP27‐S82 were evaluated by Western blotting in CAL27 and HN6 cells transfected with si‐SMAD4, si‐HSP27, scramble, ov‐SMAD4, and vector for 24 h. (G) Schematic diagram of the regulation of circGNG7 by SMAD4 binding with serine residues 78, and 82, occupying the phosphorylation site of functional protein HSP27, hindering the phosphorylation of HSP27, which reduces the HSP27‐JNK/P38 MAPK oncogenic signaling and inhibits HNSCC progression. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: RT‐PCR: reverse transcription polymerase chain reaction