Fig. 8: UBE2C is post-transcriptionally and phenotypically activated in NSCLC.

a RT-PCR and immunoblotting assay of UBE2C at both mRNA and protein levels in A549 and H1299 cells treated with miR-381 mimics or siRNA specific for ALKBH5. b Methylated RNA immunoprecipitation analysis of m⁶A levels of pre-mRNA in A549 and H1299 cells with UBE2C overexpression or ALKBH5 knockdown or in combination. c In vitro proliferation assay demonstrating that miR-381 overexpression of siRNA-specific downregulation ALKBH5 decreased cell growth and arrested UBE2C overexpression-induced cellular proliferation of both A549 and H1299. d−f Flow cytometric, immunofluorescence (Annexin V), and immunoblotting (Caspase-cleaved) assay demonstrating that miR-381 overexpression and ALKBH5 knockdown significantly induced apoptosis and reduced ectopic UBE2C expression-mediated anti-apoptosis phenotypes in A549 cells for 72 h. g Cell cycle assay indicating treatment with miR-381 mimics or siRNA-ALKBH5-induced significant G2 arrests/S reduction and reversed ectopic UBE2C expression-induced G2 reduction in A549 cells. h Colony formation assay indicating that knockdown of miR-381 or ALKBH5 in A549 cells dramatically inhibited its clonal formation and arrested UBE2C overexpression-induced enhancements in its clonogenicity. i, j RT-PCR, immunoblotting, and immunofluorescence analysis of EMT biomarkers demonstrating that miR-381 overexpression or specific knockdown of ALKBH5 activated E-cadherin and repressed vimentin and resisted overexpressing UBE2C-mediated E-cadherin repression/vimentin activation pattern in A549 cells. k Matrigel invasion assay indicating that miR-381 overexpression or ALKBH5 downregulation in A549 cells significantly decreased the cell invasive growth and partially blocked UBE2C overexpression-induced enhancement in the cell invasive growth. l Scratch assay showing forced miR-381 overexpression and siRNA-specific downregulation of ALKBH5 dramatically arrested cell migration of A549 cells with or without over expressing UBE2C. *P < 0.01, **P < 0.001, ***P < 0.0001 by Student’s t test. m RT-PCR and immunoblotting assay of endogenous ALKBH5 mRNA and protein levels in HBEC, 95-D, A549, H1299, Calu-6, H520, and PC-9 cells. n Schematic diagram depicting the epigenetic and epitranscriptional deregulation of UBE2C-autophagy repression axis that enhances NSCLC progression. As indicated in this diagram, UBE2C transcripts negatively regulated by miR-381and destabilized by the m⁶A demethylase ALKBH5 directly and selectively represses ATG3 and LC3 which further phenotypically arrest NSCLC progression