SummaryBackgroundN6-methyladenosine (m6A) is the most common and abundant mRNA modification and it plays crucial roles in many biological processes. However, as a key RNA demethylase, alkylation repair homolog protein 5 (ALKBH5) has not been well studied in human osteosarcoma. The present study sought to explore ALKBH5-mediated m6A modification and the underlying mechanisms in human osteosarcoma.MethodsThe expression of ALKBH5 and its correlation with clinicopathological features were examined by bioinformatics analysis and tissue microarrays. Cellular proliferation was detected by CCK8 assays. Cell cycle and apoptosis were analyzed by TUNEL and Flow cytometry assay. Finally, investigation of the regulatory mechanism of ALKBH5 in human osteosarcoma was performed by MeRIP assay, RNA-sequencing, dual luciferase reporter assay, RNA pull-down and RNA stability assay. Tumor xenograft models were established for in vivo experiments.FindingsOur data showed that low expression of ALKBH5 was associated with worse overall survival for osteosarcoma patients. Reducing m6A mRNA levels in human osteosarcoma cells through ALKBH5 up-regulation lead to cell proliferation inhibition, cell apoptosis and cycle arrest. We identified SOCS3, a negative regulator of STAT3, as a downstream target of ALKBH5-mediated m6A modification. And the m6A modified SOCS3 mRNA was recognized by YTHDF2, which promotes the decay of SOCS3. Mechanistically, our data revealed that ALKBH5 inactivated STAT3 pathway by increasing SOCS3 expression via an m6A-YTHDF2-dependent manner.InterpretationM6A methylation is rising as a pathway affecting tumorigenicity and tumor progression. Our findings illuminate the clinical significance of ALKBH5-mediated m6A modification in human osteosarcoma and the regulatory mechanisms underlying tumor proliferation and growth, suggesting that ALKBH5 is a potential biomarker for treatment in human osteosarcoma.FundingThis work was supported by and Science and Technology foundation of Hubei, China (Grant No.2017CFB762); the Tongji hospital foundation (Grant No.2201103013); and the National Natural Science Foudation of China (No.82002849).

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