Fig. 3

LINC00665 promotes CNBP mRNA decay by forming duplexes with 3’-UTRs via Alu elements (A) Effect of LINC00665 on the fluorescent expression of reporter gene vectors containing the CNBP mRNA 3’-UTR Alu element in 293T cells 48 h after transfection in the luciferase reporter assay. (B) RNA immunoprecipitation (RIP) was performed with a STAU1-specific antibody. The RNA was extracted, and CNBP mRNA and LINC00665 levels were evaluated by qPCR. a. The relative expression of CNBP mRNA, b. The relative expression of LINC00665. C. MS2-RIP followed by western blotting and qPCR to detect STAU1 and CNBP mRNA separately associated with LINC00665. a. Western blot for STAU1, b. qPCR for CNBP mRNA. D. MS2-RIP followed by qPCR to detect CNBP mRNA associated with LINC00665 after inhibiting the expression of STAU1. E. Expression of STAU1 and CNBP in OCSC and COC1 cells, and the effect of STAU1 inhibition on the expression of CNBP and LINC00665. a. Western blotting results after inhibition of STAU1. b. CNBP mRNA detected by qPCR. c. LINC00665 detected by qPCR. F. The stability of CNBP mRNA in OCSCs treated with shSTAU1. G. Expression of LINC00665 in each group of cells detected by qPCR after modulating CNBP expression: a. CNBP overexpression in OCSCs; b. Suppression of CNBP expression in COC1 and SKOV3 cells. H. After modulating LINC00665 expression, the expression of CNBP mRNA and CNBP protein in each group of cells was detected by qPCR and western blotting, respectively: a. qPCR assay after inhibiting LINC00665 expression in OCSC; b. qPCR assay after overexpression of LINC00665 in COC1 and SKOV3; and c. Western blot results. I. The stability of CNBP mRNA in cells transfected with the indicated vectors: a. OCSCs treated with shLINC00665, b. COC1 and c. SKOV3 ovarian cancer cells treated with LV-LINC00665, *P < 0.05