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. 2021 Oct 4;29(3):627–641. doi: 10.1038/s41418-021-00879-9

Fig. 6. THAP7-AS1/CUL4B complex initiates miR-22-3p/miR-320a -suppressed PI3K/AKT signaling.

Fig. 6

A A Venn diagram depicting five miRNAs that were upregulated by CUL4B knockdown in MKN45 cells (GSE 90503), predicted to target PIK3CA, PIK3CD and AKT3 proteins by prediction algorithms. B–D Expression of primary miR-17, miR-19, miR-22-3p, miR-320a, and miR-339 expression was assessed by RT–qPCR in MKN-45 cells respectively transfected with CUL4B (B), THAP7-AS1 (C), and SI-THAP7-AS1 (D). E–G Expression of pre-miR-320a expression was assessed by RT–qPCR in MKN-45 respectively transfected with CUL4B (E), sh-CUL4B (F), and THAP7-AS1 (G). H–K Expression of mature miR-320a and miR-22-3p expression was assessed by RT–qPCR in MKN45 respectively transfected with CUL4B (H), sh-CUL4B (I), THAP7-AS1 (J), and si-THAP7-AS1 (K). L The results indicated a decrease in luciferase activity in MKN-45 cells transfected with PGLO-AKT3-3′UTR and miR-22-3p. M–O Luciferase activity showed a decrease in MKN-45 cells transfected with PGLO-PIK3CA-3′UTR (M), PGLO-PIK3CD-3′UTR (N), PGLO-AKT3-3’UTR (O) and miR-320a. P Western blot analyses were performed to confirm the PIK3CA, PIK3CD and AKT3 expression in MKN-45 cells transfected with miR-22-3p or miR-320a mimics. Q–R Western blotting analysis of the AKT3 in MKN-45 cells overexpressing THAP7-AS1 (Q) or CUL4B (R) with or without transient transfection with miR-22-3p mimics. S–T Migration and invasion abilities of MKN-45 cells transiently transfected with indicated RNA and plasmid were observed. (×100). U–V Western blotting analysis of the PIK3CA, PIK3CD and AKT3 in MKN-45 cells overexpressing THAP7-AS1 (U) or CUL4B (V) with or without transient transfection with miR-320a mimics. W–X Migration and invasion abilities of MKN-45 cells transiently transfected with indicated RNA and plasmid were observed. (×100). *P < 0.05, **P < 0.01, ***P < 0.001.