Study On The Mechanism Of METTL3 Regulating ATAD2 To Promote The Proliferation And Invasion Of Osteosarcoma Cells

ObjectivesOsteosarcoma is the most common primary malignant bone tumor in children and young adults which is characterized by high malignancy and rapidly damage the surrounding tissues for metastasis.Early metastasis and postoperative recurrence are the main factors leading to poor prognosis in patients with osteosarcoma.Although improvement has been developed in current treatment strategies of osteosarcoma,including surgical excision combined with systemic chemotherapy or radiotherapy,the survival rate of osteosarcoma patients remains poor.For patients with localized osteosarcoma,the 5-year event-free survival(EFS)is about 70%,while the 5-year survival rate of patients with metastatic disease ranges from 15%to 30%.The treatment level of osteosarcoma has reached a plateau.Thus,it is essential to further investigate the molecular carcinogenesis of osteosarcoma and identify novel therapeutic targets for osteosarcoma treatment.RNA N6-methyladenosine(m6A)modification is one of the most abundant internal modifications in mammalian transcription mRNA,and is involved in the development of various tumors.The first reported m6A methyltransferase is Methyltransferase like protein 3(METTL3),which plays a crucial role in cancer progression.At present,whether METTL3 is involved in osteosarcoma development and its possible mechanism are not clear.In this study,we aimed to explore the role of METTL3 in the development of osteosarcoma cells and its potential mechanism.MethodsWe firstly performed immunohistochemical staining to detect the expression of METTL3 in surgically resected specimens of osteosarcoma.Then we used RT-PCR and western blot to examine the expression of METTL3 in 4 osteosarcoma cell lines,which are HOS,SAOS-2,U2OS and MG63.Immunofluorescence assay was performed to determine the localization of the METTL3 in osteosarcoma cells.Next we constructed siRNA-METTL3 to interfere the endogenous expression of METTL3 and pcdna3.1-METTL3 for overexpression to carry on further studies.By analyzing the expression of METTL3 mRNA and protein expression,the validity of siRNA-METTL3 and pcdna3.1-METTL3 was determined.The m6A RNA methylation level when METTL3 is knock down or over expressed was further detected.CCK8 assay and cell clone assay were performed to determine the effect of METTL3 on the proliferation and colony forming ability of osteosarcoma cells.Next,we performed Transwell assay to determine the effect of METTL3 on the migration and invasion of osteosarcoma cells.Flow cytometry was used to analyze the apoptosis of osteosarcoma cells in the case of METTL3 knockdown or overexpression.In order to further study the molecular mechanism of METTL3 participating in these cell processes,we performed Transcriptome sequencing(RNA-seq)technology to analyze the change of mRNA transcription level after METTL3 knockdown in MG63 cells.We selected ATAD2 as the downstream target of METTL3 for further study.The loss and gain of function assays were performed to investigate the biological role of ATAD2 in osteosarcoma cellsIn order to study the mechanism of METTL3 regulating ATAD2,we performed RNA m6A methylation sequencing(m6A-seq)on MG63 cells to analyze the changes of m6A mRNA methylation level after METTL3 knockdown and explore the mechanism of METTL3 regulating ATAD2.ResultsThe results of immunohistochemical staining showed that the expression of METTL3 in osteosarcoma was significantly higher than that in adjacent tissues.The METTL3 expression of U2OS cells was the lowest in both mRNA and protein levels,while relatively higher in SAOS-2 and MG63 cells.U2OS,SAOS-2 and MG63 cells were used for the next experiments.Immunofluorescence assays showed that METTL3 localized in both cytoplasm and nucleus of osteosarcoma cells.To investigate the functional role of METTL3 in osteosarcoma cells,the expression of METTL3 was knocked down by siRNA-METTL3 interference at mRNA and protein level in SAOS-2 and MG63 cells,while the expression was obviously up-regulated by transfection with pcDNA3.1-METTL3 in U2OS cells.The m6A RNA methylation level showed a significant decrease in both SAOS-2 and MG63 cells,whereas up-regulation of METTL3 increased the m6A RNA methylation level in U2OS cells.A significant decrease in cell viability was observed in METTL3-inhibited SAOS-2 cells.Similarly,METTL3 knockdown also inhibited the viability of MG63 cells.However,up-regulation of METTL3 had no significant effect on the viability of U2OS cells.Consistent with CCK8 assay,SAOS-2 and MG63 cells colony-forming abilities were also suppressed by knocking down METTL3 compared with the corresponding control groups,but the colony-forming ability of U2OS cells was still not affected by METTL3 overexpression.METTL3 knockdown resulted in a significant depression in the migration ability of SAOS-2 and MG63 cells compared to the corresponding control groups.Up-regulation of METTL3 still had no effect on the migration ability of U2OS cells.Similarly,the invasion abilities of SAOS-2 and MG63 cells were also suppressed after METTL3 inhibition,but the invasion ability of U2OS cells was not impaired after METTL3 overexpression.We found that there was a significant increase in the percentage of apoptotic cells of METTL3 knockdown cells compared with the control cells.However,the apoptosis of U2OS cells was not impaired by METTL3 overexpression.The expression of anti-apoptotic protein Bcl-2 was significantly down-regulated by METTL3 knockdown in both SAOS-2 and MG63 cells,while the expression of pro-apoptotic proteins Bax and cleaved Caspase 3 was obviously up-regulated.Obviously,the up-regulation of METTL3 had no significant effect on the expression of these proteins in U2OS cells.RNA-seq showed that there were 578 genes were up-regulated in METTL3 knockdown MG63 cells,and 481 genes was down-regulated.Different gene expression patterns were observed between METTL3 knockdown cells and negative control cells.In consistent with RNA-seq,RT-PCR and Western blot analysis revealed that METTL3 knockdown significantly inhibited the protein expression of ATPase family AAA domain-containing protein(ATAD2)in both SAOS-2 and MG63 cells Knockdown of ATAD2 significantly inhibited the proliferation of both SAOS-2 and MG63 cells.On the contrary,overexpression of ATAD2 promoted the proliferation of SAOS-2 and MG63 cells.Transwell assay revealed that knockdown of ATAD2 decreased the invasion abilities of both SAOS-2 and MG63 cells,whereas up-regulation of ATAD2 resulted in an increase in the invasion abilities of both cell lines.METTL3 knockdown could reverse the promotion of ATAD2 overexpression on proliferation and invasion of osteosarcoma cells.m6A-seq in MG63 cells showed that the methylation level of ATAD2 mRNA decreased after METTL3 knockdown,suggesting that METTL3 may promote the proliferation and invasion of osteosarcoma cells by regulating ATAD2 through m6A modification.Conclusion1.METTL3 is distributed in both nucleus and cytoplasm of osteosarcoma cells;2.Knockdown METTL3 suppresses the migration and invasion of osteosarcoma cells;3.Knockdown METTL3 may promote apoptosis in osteosarcoma cells by regulating the Bcl-2/Bax axis and Caspase cascade;4.ATAD2 promotes the proliferation and invasion of osteosarcoma cells;5.METTL3 may regulate ATAD2 by m6A modification to promote the proliferation and invasion of osteosarcoma cells.