Fig. 4

TRAF3IP2-AS1 down-regulates TRAF3IP2 by recruiting HNRNPK to TRAF3IP2 promoter. A-B The mRNA and protein levels of TRAF3IP2 were detected in UOK109 cells transfected with dCas9 and guide RNA targeting TRAF3IP2-AS1 promoter (gTRAF3IP2-AS1) and 786-O cells transfected with siRNA and antisense oligonucleotides (siTRAF3IP2-AS1). C UOK109 cells were transfected with MS2-GFP and TRAF3IP2-AS1-12 × MS2, then DNA region binding to TRAF3IP2-AS1 was enriched by anti-GFP antibody. D UOK109 cells were transfected with dCas9-Flag and gTRAF3IP2, then dCas9-ChIP assay was performed with anti-Flag antibody. E The mRNA level of TRAF3IP2 was detected by qRT-PCR after transfected with shRNA targeted the potential TRAF3IP2-AS1 binding proteins. F UOK109 cells were transfected with dCas9-Flag and gTRAF3IP2, then the proteins binding to the promoter region of TRAF3IP2 was enriched by anti-Flag antibody. G UOK109 cells were lysed, then ChIP and RIP were performed with anti-HNRNPK antibody. H UOK109 cells and 786-O cells were transfected with dCas9-Flag, gTRAF3IP2 and indicated lentivirus, then the proteins binding to the promoter region of TRAF3IP2 was enriched by anti-Flag antibody. I HEK293T cells were co-transfected with TRAF3IP2-AS1 truncations and MS2-GFP, then MS2-RIP assay was performed. J HEK293T cells were transfected with HNRNPK truncations, then RIP assay was performed. K-M The mRNA and protein levels of TRAF3IP2 and HNRNPK were detected in UOK109 cells and 786-O cells transfected shHNRNPK. N Schematic illustration of dCas9-based system to deliver the exogenous expressed protein to the promoter of TRAF3IP2. O-Q The mRNA and protein levels of TRAF3IP2 were detected in UOK109 cells and 786-O cells transfected dCas9-HNRNPK and gTRAF3IP2. The data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001