WTAP Promotes Osteosarcoma Progression via Regulating the m6A Methylation of HMBOX1

Background: N6-methyladenosine (m6A) regulators are involved in the progression of various cancers via regulating m6A modification. However, the potential role and mechanism of the m6A modification in osteosarcoma (OS) remains obscure. Methods: The expression of m6A regulators were detected using qPCR and western blot. Cell proliferation, colony formation, wound healing and transwell invasion assays were used to reveal the role of WTAP in the progression of OS. The mechanism of WTAP-mediated m6A modification was explored using M6A dot blot, RNA-seq and MeRIP-seq, MeRIP-qRT-PCR and luciferase reporter assays. Results: Here, WTAP was found to be high expression in OS tissue and it was an independent prognostic factor for OS patients . Functionally, WTAP, as an oncogene, was involved in the proliferation and metastasis of OS. Mechanistically, M6A dot blot, RNA-seq and MeRIP-seq, MeRIP-qRT-PCR and luciferase reporter assays showed that HMBOX1 is the target gene of WTAP, which regulated HMBOX1 stability in an m6A-dependent manner by targeting to the 3’UTR of HMBOX1 mRNA. In addition, HMBOX1 expression was downregulated in OS and negatively linked to WTAP expression in OS patients. Silenced HMBOX1 evidently attenuated shWTAP-mediated suppression on OS growth and metastasis in vivo and vitro. Finally, WTAP/HMBOX1 regulated OS growth and metastasis via PI3K/AKT pathway. Interpretation: The study demonstrates the critical role of the WTAP-mediated m6A modification in the progression of OS, which could provide novel insights into therapy of OS. Funding Statement: This study was supported by National Natural Science Foundation of China (Grant Nos. 81772866); National Natural Science Foundation of China (Grant Nos. 81502331); Natural Science Foundation of Hunan Province (Grant Nos. 2018JJ2617); Natural Science Foundation of Hunan Province (Grant Nos. 2016JJ3176); National Key Research and Development Program of China (No. 2016YFC1201800) ; Natural Science Foundation of Hunan Province (No. 2018SK2090). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Informed consent was obtained from each patient or their guardians, and the study were approved by the Ethics Committee of The Third Xiangya Hospital of Central South University. All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of The Third Xiangya Hospital of Central South University, Changsha, China.