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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Long non-coding RNA ZEB1-AS1 regulates miR-200b/FSCN1 signaling and enhances migration and invasion induced by TGF-β1 in bladder cancer cells

Fig. 4

ZEB1-AS1 downregulates miR-200b, upregulates fascin-1 (FSCN1), and promotes cell migration and invasion. a Luciferase assays were performed in T24, RT4, and 293 T cells co-transfected for 24 h with miR-negative control (NC) or miR-200b and a plasmid containing wild-type or mutant-type ZEB1-AS1 3′untranslated region (UTR) upstream the luciferase gene. Firefly luciferase activity of each sample was normalized by Renilla luciferase activity. Data were analyzed by T-test. b RNA-binding protein immunoprecipitation (RIP) assays with anti-AGO2 antibodies were performed in T24 and RT4 cells transiently transfected with miR-200b; ZEB1-AS1 levels were detected by quantitative PCR (qPCR); 10% input was used as positive control and RIP with anti-IgG antibodies served as negative control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. Data were analyzed by T-test. c Transfection efficiency of ZEB1-AS1 knockdown (si1, si2) and overexpression (OE) in T24 and RT4 cells was detected by qPCR. Data were analyzed by T-test. d and e The levels of miR-200b (d) and FSCN1 (e) were measured by qPCR after ZEB1-AS1 knocked down or overexpressed in T24 and RT4 cells. Data were analyzed by T-test. f E-cadherin, N-cadherin, vimentin, and FSCN1 protein expression in T24 and RT4 cells in which ZEB1-AS1 had been knocked down or overexpressed. Data were analyzed by T-test. (g) FSCN1 levels in T24 cells co-transfected with miR-NC or miR-200b and with an empty vector (EV) or a plasmid overexpressing ZEB1-AS1. Data were analyzed by T-test. h and i Transwell assays (without or with Matrigel) to detect cell migration (h) and invasion (i) of T24 and RT4 after ZEB1-AS1 silencing or overexpression. j and k Transwell assays (without or with Matrigel) to detect cell migration (j) and invasion (k) of T24 and RT4 co-transfected with miR-NC or miR-200b and with an EV or a plasmid overexpressing ZEB1-AS1. Data were analyzed by T-test. Data are presented as the mean ± standard deviation (SD). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

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