Fig. 6
ALKBH5 decreases YAP activity by regulating miR-107/LATS2 in an HuR-dependent manner. (a) The positive correlation between ALKBH5 and HuR analyzed from TCGA database. (b) Bioinformatics prediction the interaction between ALKBH5 and HuR (https://thebiogrid.org/). (c) The protein levels of ALKBH5 and HuR was analyzed by western blot assay. (d) Co-IPs performed using lysates collected from A549 cells with immunoprecipitation by either ALKBH5 or HuR antibodies. (e-g) A549 cells were transfected into indicated genes. The RNA levels of HuR, ALKBH5 and miR-107 were analyzed by qPCR and RT-PCR. (h) Putative miR-107 binding sites in the 3′UTR sequences of LAST2. (i) The luciferase reporter activity of the wild-type and mutated LATS2 was detected in A549 cells. (j) MS2-RIP followed by qPCR to measure miR-107 associated with 3′UTR of LATS2 in A549 and H1299 cells. (k) qPCR analyzed the RNA levels of miR-107 and LATS2 3’UTR in the products of A549 and H1299 cells with transfection with indicated genes by pulldown with biotin. (l, m) A549 cells were transfected into indicated genes. The RNA and protein levels of miR-107, LAST2, YAP and p-YAP were analyzed by RT-PCR and western blot assays. (n) Subcellular localization of YAP, CTGF and Cyr61 were examined in A549 cells determined by western blot assay. (o-r) A549 cells were transfected with indicated genes of ALKBH5 and miR-107 mimics (miR-107 m). (o) The protein levels of CTGF and Cyr61 were analyzed by western blot assay. (p) The cellular viability and growth were analyzed by CCK8 assay. (q) The cellular invasion and migration growths were analyzed by transwell. (r) The expressions of E-cadherin and Vimentin were analyzed by qPCR assay. Results were presented as mean ± SD of three independent experiments. **P < 0.01 indicates a significant difference between the indicated groups. ns, not significant