Fig. 8. IGF2BPs were required for the ALKBH5-induced regulation of mRNA expression and cell proliferation.

A Expression levels of IGF2BP1, IGF2BP2, and IGF2BP3 (IGF2BPs) protein determined using western blot analysis were compared between cell lines. B IGF2BP protein levels in cells transfected with siALKBH5 (left end), those in cells transfected with siIGF2BP1, siIGF2BP2, or siIGF2BP3 with or without siALKBH5 (middle two lanes), or those with siNC (right end indicating both siALKBH5 and siIGF2BPs were negative) were confirmed via western blot analysis. C Relative mRNA expression levels of CDKN1A in PC9 cells or those of TIMP3 in A549 cells transfected with siALKBH5 were analyzed via qPCR and compared with those in cells cotransfected with siALKBH5 and one of the siIGF2BPs. Gene expression was normalized to the GAPDH expression and was shown relative to the expression in siNC (n = 3). D Expression levels of YTHDF2 protein determined using western blot analysis were compared between cell lines. E Relative mRNA expression levels of CDKN1A in PC9 cells or those of TIMP3 in A549 cells transfected with siALKBH5 were analyzed via qPCR and compared with those in cells cotransfected with siALKBH5 and siYTHDF2. Gene expression was normalized to the GAPDH expression and was shown relative to the expression in siNC (n = 3). F The remaining RNA level of CDKN1A in PC9 cells or of TIMP3 in A549 cells after actinomycin D treatment for 0, 2, 4, and 6 h was determined using qPCR and normalized to the expression at 0 h. RNA decay rate in cells transfected with siALKBH5 and/or one of the siIGF2BPs and siNC were compared with the stability of CDKN1A and TIMPs (n = 3). G Cell proliferation relative to baseline in PC9 and A549cells transfected with siALKBH5 was assessed via the CCK-8 assay and compared with that in cells cotransfected with siALKBH5 and one of the siIGF2BPs (n = 3). H Schematic illustration for the proposed mechanism of tumorigenicity via ALKBH5 in non-small-cell lung cancer. Upregulation of ALKBH5 in NSCLC reduces m⁶A modifications on the 3′ UTR of specific genes. The loss of m⁶A decreases the opportunity for recognition by IGF2BPs and destabilizes the target transcripts such as CDKN1A (p21) and TIMP3. Downregulation of CDKN1A (p21) and TIMP3 induces cell cycle alteration and inhibits apoptosis. This ALKBH5–IGF2BPs axis promotes cell proliferation and tumorigenicity, which, in turn, causes the unfavorable prognosis of NSCLC. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, ^***P < 0.001, ^****P < 0.0001 indicates a significant difference between the indicated groups.