Fig. 5

RP11-757G1.5 sponges miR-139-5p and modulate YAP1 expression. a Subcellular localization of RP11-757G1.5 was determined by RT-qPCR in HCT-116 and SW480 cell lines. b miR-139-5p-binding sequence in RP11-757G1.5 and YAP1 3’UTR. A mutation was generated in RP11-757G1.5 in the complementary site for miR-139-5p binding. c Luciferase activity of a luciferase reporter plasmid (pLuc) containing wild-type or mutant RP11-757G1.5 co-transfected with miR-139-5p was determined using the dual luciferase assay. d MiR-139-5p and pLuc plasmid containing YAP1 3’UTRs were co-transfected with pcDNA3.1-757G1.5 or empty vector into HCT-116 cells to verify whether RP11-757G1.5 can function as a ceRNA of miR-139-5p. e Cellular lysates from HCT-116 cells were used for RIP with an anti-Ago2 antibody or IgG antibody. The levels of RP11-757G1.5 and miR-139-5p were detected by RT-qPCR. f and l The expression levels of YAP1 in HCT-116 cells transfected with pcDNA3.1-757G1.5 and SW480 cells transfected with sh-757G1.5 were analyzed by RT-qPCR and western blotting. g The elevated expression of YAP1 in tissue level was detected by immunohistochemistry (IHC) test, normalized to para-tumor tissue group. Scale bar, 200 μm; 50 μm. h YAP1 was up-regulated in CRC tissue and cell lines as determined by a RT-qPCR, normalized to para-tumor tissue group and NCM460 group, respectively. i-k Correlation between RP11-757G1.5, miR-139-5p, and YAP1 expression in CRC and normal colon specimens as detected by RT-qPCR (n = 41). m Western blot assays were performed to test YAP1 expression after HCT-116 cells were transfected with miR-139-5p mimic or co-transfected with miR-139-5p mimic and pcDNA3.1-757G1.5. Meanwhile, SW480 cells were transfected with miR-139-5p inhibitor or co-transfected with miR-139-5p inhibitor and sh-757G1.5#1. Data from western blot assay has been represented as a quantification graph normalized to the levels of GAPDH together with the statistical tests. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test. Data are representative of at least three independent experiments