Mechanism Of RNA Demethylase ALKBH5 Inhibiting Malignant Phenotype Of Osteosarcoma

Background and ObjectiveOsteosarcoma is the most common primary malignant bone tumor in children and adolescents.It mainly grows on the metaphysis of long bones.It is characterized by the production of the bone-like matrix.It has a high recurrence rate,multi-drug resistance,and early lung cancer.The characteristics of the transfer.For patients with osteosarcoma,the combination of surgical treatment and chemotherapy is still the traditional treatment method.Although the diagnosis and treatment of osteosarcoma have made great progress in the past ten years,60%-70% of osteosarcoma patients still cannot prolong their survival after treatment,especially those with distant metastasis and recurrence in the lungs.The 5-year survival rate is only about 20%.Therefore,how to improve the therapeutic effect of osteosarcoma is an urgent problem to be solved.The search for effective molecular targets is extremely important for the biological treatment of osteosarcoma.Various studies have shown that m6 A is a widespread epigenetic modification on RNA molecules,and it participates in various stages of the RNA life cycle,such as transcription,processing,translation,and metabolism.RNA demethylase ALKBH5 is an important enzyme that can change the level of m6 A modification,affect the function of RNA,and play an important role in the development and development of diseases.Studies have reported that ALKBH5 plays an important role in pancreatic cancer,lung cancer,and liver cancer.However,it is unclear whether ALKBH5 is involved in the occurrence and development of osteosarcoma.The purpose of this study is to clarify the expression of RNA demethylase ALKBH5 in osteosarcoma and to preliminarily study its mechanism of influence on the growth and metastasis of osteosarcoma.Method:1.Comprehensive analysis of the expression of ALKBH5 in osteosarcoma tissue by immunohistochemistry,fluorescence quantitative PCR,and western blotting.Using m6 A quantitative detection kit to detect m6 A levels in osteosarcoma tissues and cells.2.Transfect the ALKBH5 overexpression plasmid into the osteosarcoma cell line transiently,and detect the change of ALKBH5 expression by RT-q PCR and Western blot.The Ed U experiment and CCK8 experiment were used to observe the changes in cell growth ability after ALKBH5 overexpression,and the changes in the cell cycle were detected by flow cytometry.Next,the effect of overexpression of ALKBH5 on cell invasion and migration was observed through the scratch healing experiment and the Transwell experiment.Finally,we used the stably transfected Vector/U2 OS and ALKBH5/U2 OS osteosarcoma cell lines to establish a nude mouse subcutaneous tumor model of human osteosarcoma and observe the formation of the tumor.3.After changing the expression of ALKBH5,Western blot was used to detect the expression of EMT-related proteins and the core protein of the Wnt/β-catenin pathway.Result:1.Compared with neighboring tissues,ALKBH5 has a lower expression in osteosarcoma tissue,and the level of m6 A in osteosarcoma tissue is higher.Compared with normal osteoblasts,the expression level of ALKBH5 in the osteosarcoma cell line is lower,while the level of m6 A is higher.ALKBH5 can negatively regulate the m6 A level of osteosarcoma.2.After overexpression of ALKBH5 in the osteosarcoma cell lines U2 OS and MG63,CCK8 and Ed U experiments showed that its growth ability was weakened,and the cell cycle was blocked at the G1 phase;scratch healing and Transwell experiments showed that its invasion and migration ability was also significantly inhibited;In vivo experiments also confirmed that the tumor size and growth rate of nude mice after increasing the level of ALKBH5 were not as good as those of the control group.3.After osteosarcoma cells overexpress ALKBH5,the expression of Wnt3,β-catenin and LEF1 protein decrease.The Wnt/β-catenin pathway inhibits the epithelial-to-mesenchymal transition(EMT)process of osteosarcoma cells to inhibit invasion and migration.Conclusion:The RNA demethylase ALKBH5 is down-regulated in osteosarcoma.It can be used as a tumor suppressor gene to inhibit the growth and metastasis of osteosarcoma,and inhibit the epithelial-mesenchymal transition(EMT)process of osteosarcoma cells through the Wnt /β-catenin pathway Then inhibit invasion and migration.The results provide a new theoretical basis for studying the growth and metastasis mechanism of osteosarcoma and will provide new research directions for targeted therapy of osteosarcoma.