Fig 2: ALYREF promotes glycolysis and proliferation of BCa cells. (A) There are 569 genes significantly changed in ALYREF knockdown (shALYREF) compared to control (shNC) 5637 cells. The results were determined by RNA-seq and shown by the volcano plot. (B) GSEA showes that the glycolysis set is strongly correlated with ALYREF. Significantly regulated genes are also presented. (C) A Venn diagram showes 206 overlapping genes between RNA-seq data from the present study and ALYREF RIP-seq data from Yang et al. [41]. (D, E, F) Effects of ALYREF knockdown, ALYREF wild-type overexpression, or ALYREF mutation overexpression on glucose utilization, lactic acid production, and intracellular ATP level in 5637 and T24 cells. (G) Effects of ALYREF knockdown, ALYREF wild-type overexpression, or ALYREF mutation overexpression on cell proliferation detected by CCK-8 assays. (H) The efficiencies of cell colony formation in BCa cells with ALYREF knockdown. (I) Effects of ALYREF wild-type or ALYREF mutation overexpression on cell proliferation measured by CCK-8 assays. (J, K, L) Effects of ALYREF knockdown or overexpression on tumor volume and weight in nude mouse xenograft models. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: shALYREF, knockdown of Aly/REF export factor; NC, negative control; shNC, negative control of knockdown; ES, enrichment score; FDR, false discovery rate; GPI, glucose-6-phosphate isomerase; PPP2R1A, protein phosphatase 2 scaffold subunit Aalpha; ALDOA, aldolase, fructose-bisphosphate A; TPI1, triosephosphate isomerase 1; ENO1, enolase 1; PFKL, phosphofructokinase, liver type; PKM2, Pyruvate kinase muscle isozyme M2; RIP-seq, RNA Iimmunoprecipitation sequencing; ATP, adenosine triphosphate; ALY-WT, Aly/REF export factor wild type; ALY-MUT, Aly/REF export factor mutation; ALY-OE, Aly/REF export factor overexpression; CCK-8 assay, cell counting kit-8 assay; BCa, bladder cancer.

Fig 3: Overexpression of PKM2 reverses the inhibition of aerobic glycolysis and cell proliferation caused by ALYREF knockdown in BCa cells. (A) Western blotting of PKM2 expression in 5637 and T24 cells co-transfected with lentivirus vector (ALYREF knockdown or control) and plasmid vector (PKM2 or control). (B) Glucose assay, lactic acid assay, and ATP assay showe that overexpression of PKM2 rescues inhibition of aerobic glycolysis caused by ALYREF knockdown in 5637 and T24 cells. (C) Overexpression of PKM2 rescues the cell proliferation affected by ALYREF knockdown in 5637 and T24 cells by CCK-8 assays. (D) Overexpression of PKM2 rescues the colony formation affected by ALYREF knockdown in 5637 and T24 cells by colony formation assays. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: PKM2, Pyruvate kinase muscle isozyme M2; GFP, green fluorescent protein; shNC, negative control of knockdown; ALYREF, Aly/REF export factor; shALYREF, knockdown of Aly/REF export factor; BCa, bladder cancer.

Fig 4: HIF-1a up-regulates ALYREF expressions in BCa cells. (A) Bioinformatic analysis identifies HIF-1a as the potential transcription factor and ‘ACGTGC’ as the consensus hypoxia response element for ALYREF. (B) ALYREF and PKM2 mRNA levels are up-regulated in 1% O2 group as compared with 20% O2 group by qRT-PCR. 5637 and T24 cells were exposed to 20% or 1% O2 for 24 h. (C) HIF-1a, ALYREF, and PKM2 proteins are up-regulated in 5637 and T24 cells exposed to 1% O2 as compared with those exposed to 20% O2 for 48 h by Western blotting. (D) Relative luciferase activity was measured in 293T cells transfected with reporter plasmids containing either wild-type or mutant ALYREF promoter fragments. Then cells were exposed to 20% or 1% O2 for 48 h. (E) Relative luciferase activity was measured in 293T cells transfected with reporter plasmids containing PKM2 promoter fragments. Then cells were exposed to 20% or 1% O2 for 48 h. The ratio of luciferase activity was determined. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: HRE, hypoxia-responsive element. Abbreviations: BCa, bladder cancer; HIF-1a, hypoxia- inducible factor- 1-alpha; ALYREF, Aly/REF export factor; PKM2, Pyruvate kinase muscle isozyme M2; Wt, wild type; Mut, mutation; ALY-WT, Aly/REF export factor wild type; ALY-MUT, Aly/REF export factor mutation; hLuc, fireflyer luciferase; Rluc, Renillar luciferase.

Fig 5: Working model: The role of HIF-1a/ALYREF/PKM2 axis in BCa. Abbreviations: NSUN2, NOP2/Sun RNA methyltransferase family member 2; m5C, 5-methylcytidine; me, methyl; BCa, bladder cancer.