Fig. 7

Enz differentially regulates AR binding on the lncRNA-p21 promoter region and promotes lncRNA-p21 expression. a The qPCR analysis of lncRNA-p21 expression in C4-2 and CWR22RV1 (22RV1) cells w/wo AR knockdown. b The correlation of AR and lncRNA-p21 expressions in the human PCa samples (n = 20). c The schematic depiction of putative androgen response elements (AREs) on lncRNA-p21 3 kb promoter region. d ChIP assays to identify the AR binding in the putative AREs w/wo Enz treatment. The C4-2 cells were treated w/o Enz for 3 days, and then the AR binding to the putative AREs on the lncRNA-p21 promoter was analyzed by qPCR. e, f ChIP assays to identify (e) H3K4me3 enrichment and (f) FOXA1 binding to the putative ARE regions on the lncRNA-p21 promoter. g Luciferase assay to identify the transactivation of lncRNA-p21 3 kb promoter region after different anti-androgen treatments. h Luciferase assay to identify the transactivation of lncRNA-p21 3 kb promoter region in C4-2 cells with pLKO or shAR (left) and in PC3 cells with pWPI or OE-AR (right). (*p < 0.05, **p < 0.005). i The Enz treatment vs DMSO control effects on mutated AGRE (AGRE-mut) and mutated ARE5 (ARE5-mut) vs wild type (WT) lncRNA-p21 promoter transactivation were analyzed by luciferase assay in C4-2 cells. j The qPCR to detect the levelS of lncRNA-p21 and NE markers in C4-2 pLKO and shSP1 cells w/o Enz treatment. k C4-2 pLKO and shSP1 cells were treated w/o Enz, and then the lncRNA-p21 promoter transactivation activity was analyzed by luciferase assay. l ChIP assay to identify the SP1 binding on the ARE5 region before and after Enz treatment. m The SP1RE cluster on lncRNA-p21 promoter region was deleted and constructed into pGL3 luciferase reporter plasmid. Enz effects on WT-lncRNA-p21 promoter or SP1RE deleted-lncRNA-p21 promoter transactivation were analyzed by luciferase assay. For i, k, the data are presented as mean ± SD, *p < 0.05, **p < 0.005, N.S. not significant by t test for two groups or ANOVA for more than two groups