Fig. 5
GATA6-AS1 increases SNAI1 mRNAs degradation via repressing FTO expression. A RT-qPCR and western blotting analysis of SNAI1 expression in PDAC cells after transfection with the indicated vector under 1% O2. B RIP assays were performed in Panc-1 cells to search the possible upstream m6A enzymes (METTL3, METTL14, WTAP, ALKBH5 and FTO), which might bind with GATA6-AS1. C RNA pull-down and western blot assays were performed to confirm the association between FTO and GATA6-AS1. D RNA pull-down using sequentially deleted GATA6-AS1 fragments demonstrates the binding segment of GATA6-AS1 with FTO. E, F RT-qPCR and western blotting analysis of SNAI1 expression in PDAC cells with transfection of the indicated vector under 1% or 20% O2. G RNA stability assays analysis of SNAI1 mRNA in PDAC cells transfection with indicated vectors and treatment with Actinomycin D. H–J Me-RIP and qPCR assay analysis of the relative m6A enrichment of SNAI1 mRNA in PDAC cell lines with transfection of the indicated vectors under 1% or 20% O2 conditions. K MeRIP combined with RT-qPCR analysis of the relative m6A enrichment at three sites of SNAI1 transcript predicted by the online tools (SRAMP) in PDAC cell lines. L The schematic illustration was established for the luciferase reporter. Different from the wild-type group, two adenosines were replaced by cytosines in mutant group. M Wild-type or mutant SNAI1 of luciferase reporters were transfected into PDAC cells (with the shRNA vectors or controls), followed by the measurement of luciferase activity. NC negative control; Data are shown as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001