m⁶A Demethylase ALKBH5 Promotes Tumor Cell Proliferation by Destabilizing IGF2BPs Target Genes and Worsens the Prognosis of Patients with Non-Small Cell Lung Cancer
2021
Background: The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small cell lung cancer (NSCLC) development remains to be completely elucidated. Methods: The current study investigated the involvement of ALKBH5 in NSCLC development using immunostaining of clinical NSCLC specimens as well as cancer-related cellular functions (cell proliferation, migration ability, cell cycle, and apoptosis) in ALKBH5-knockdown lung cancer cell lines. Moreover, a microarray was utilized to comprehensively analyze mRNA and m6A in ALKBH5-knockdown cells. m6A target genes were identified using the methylated RNA immunoprecipitation (MeRIP) assay with m6A antibody. Furthermore, mRNA stability and protein expression owing to m6A modification (the target genes) were examined. Results: Clinicopathological analysis revealed that increased ALKBH5 expression was an independent prognostic factor associated with unfavorable overall survival in NSCLC (hazard ratios, 1.468; 95% confidence interval, 1.039—2.073). In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines BEAS2B and HEK293. Comprehensive analysis of microarray and MeRIP quantitative-polymerase chain reaction revealed that 3′ untranslated regions (3′ UTRs) of CDKN1A, TIMP3, E2F1, and CCNG2 mRNA were potential targets of ALKBH5. Depending on the lung cancer cell lines, increased expression of CDKN1A or TIMP3 and decreased cell proliferation were observed by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. Conclusions: Upregulation of ALKBH5 in NSCLC reduces m6A modifications on the 3′ UTR of specific genes. Loss of m6A causes a decrease in opportunity for recognition by IGF2BPs and destabilizes the target transcript, such as CDKN1A (p21) and TIMP3. Downregulation of CDKN1A (p21) and TIMP3 induces cell cycle alteration and inhibits apoptosis. The ALKBH5—IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.
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