Fig. 5
CircFGFR1int2 facilitated FGFR1 transcription by recruiting FUS/P65. A Bioinformatics analyses by RNAhybrid showed multiple potential circFGFR1int2 binding sites (green boxes) enriched around P65 binding site (dotted line box) in the FGFR1 promoter. B Chromatin isolation by RNA purification (ChIRP) followed by Western blot analysis and PCR revealed that FUS, P65, circFGFR1int2, and FGFR1 promoter sequences containing the P65 binding site were enriched in the complexes obtained with biotin-labelled-circFGFR1int2 probes, but not by random-sequence control RNA probe. Knockdown of circFGFR1int2 by ASO-circFGFR1int2 decreased recovery of the above components. C ChIP with anti-FUS or anti-P65 showed that knockdown of circFGFR1int2 by ASO-circFGFR1int2 significantly reduced retrieval of FGFR1 promoter sequence containing the P65 binding site (lower represented qRT-PCR analysis). D Upper: dual-luciferase reporter constructs with wildtype FGFR1 promoter containing P65 binding site (pGL3-P691), or truncated sequence (pGL3-P500) or mutated binding sites (pGL3-P691-P65 MUT, pGL3-P691-circFGFR1int2 MUT). Lower: dual-luciferase reporter assays showed significant FGFR1 promoter activity containing P65 binding site (pGL3-P691), which was significantly reduced when the P65 binding site was removed by truncation (pGL3-P500), or when P65 or circFGFR1int2 binding sites were mutated (pGL3-P691-P65 MUT and pGL3-P691-circFGFR1int2 MUT). Knockdown of circFGFR1int2 by ASO-circFGFR1int2 significantly decreased the FGFR1 promoter activity, while artificial overexpression of circFGFR1int2 by OE-circFGFR1int2 rescued the FGFR1 expression. E, F CircFGFR1int2 knockdown by ASO-circFGFR1int2 had no significant effects per se on P65 and FUS mRNA and protein expressions. Error bars for ChIRP, ChIP, Dual-luciferase reporter assays, qRT-PCR, and Western blot assays represented mean ± standard deviation (SD) from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant