Analysis And Identification Of M~6A Methylation Regulators In Metastatic Osteosarcoma

Objective Osteosarcoma is the most common primary bone malignant tumor in children and adolescents,characterized by rapid growth and early metastasis.Distant metastasis is the most common and the most important fatal factor in osteosarcoma patients,especially lung metastasis.Therefore,lung metastasis is the primary problem in the treatment of osteosarcoma.Thus,the identification of biomarkers significantly associated with metastasis is of positive significance to accurately predict the risk of metastasis or progression of osteosarcoma,better understand the metastatic mechanism of osteosarcoma,and further search therapeutic targets of osteosarcoma.Methods 1.RNA sequencing transcriptome data of 88 patients with osteosarcoma and corresponding clinicopathological information were downloaded from the TARGET database.Combined with published literature information,21 RNA m6 A methylation regulators,including eight methyltransferases(METTL3,METTL14,WTAP,RBM15,KIAA1429,RBM15 B,CBLL1,ZC3H13),two demethylation enzymes(ALKBH5,FTO),11m6 A binding proteins(HNRNPA2B1,HNRNPC,YTHDF1,YTHDF2,YTHDF3,ELAVL1,YTHDC1,YTHDC2,IGF2BP1,FMR1,LRPPPC),were studied.Protein-protein interactions were analyzed in 21 m6 A methylation regulators using the protein interaction database STRING.The "cluster Profiler" package was used for gene functional enrichment analysis in R.The "go-plot" visualization package was used to describe the enrichment of 21 methylation regulators and their functional categories in R.LASSO Cox regression was used to screen the key genes of m6 A methylation regulators in osteosarcoma.Kaplan Meier analysis was used to compare the difference in overall survival between the high-risk and low-risk groups.Using the "PHat Map" package,heat maps were drawn in R.Methylation sites of RBM15 in different osteosarcoma cell lines were evaluated using the Cancer Cell Line Encyclopedia database.The Oncomine database was used to further detect the expression levels of selected m6 A RNA methylation regulators in the samples of recurrent tumors and different subtypes of osteosarcoma.Score data of 6 immune infiltrating cells in sarcomas were obtained from the Tumor Immune Evaluation Resource Database.2.The expression level of RBM15 m RNA in osteosarcoma cell lines was detected by the quantitative real-time PCR(RT-PCR).Western Blot was used to detect the expression of RBM15 protein in osteosarcoma cell lines.Immunohistochemistry and Western Blot were used to confirm the difference in RBM15 expression between primary osteosarcoma and metastatic osteosarcoma of the lung.And the m RNA expression of RBM15 was detected in primary osteosarcoma tissues and metastatic osteosarcoma tissues by the quantitative real-time PCR.3.The si RNA targeted RBM15 was transfected into the osteosarcoma cell lines with high metastatic potential(MNNG and 143B).The effect of RBM15 interference was evaluated by RT-PCR analysis.After confirming the success of interference,the changes in osteosarcoma cell proliferation,colonization,invasion,and metastasis were analyzed by CCK8,plate cloning experiment,Transwell,and scratch experiment,respectively.4.The MNNG and HOS cells were used to construct stable cell lines with RBM15 knockdown and overexpression by lentivirus transfection,respectively.The functional effects of RBM15 on the colonization,invasion,and metastasis of osteosarcoma cells were analyzed by CCK8,plate cloning assay,Transwell,and scratch assay,respectively.The tumorigenesis,invasion,and metastasis of osteosarcoma cell lines after RBM15 knockdown were evaluated in nude mice by subcutaneous tumorigenesis and lung metastasis models.5.Using Next-generation sequencing technology,the differential expression genes were detected and obtained in RBM15 deleted or overexpressed cells.Bioinformatics analyses were carried out to analyze the cellular functions and related signaling pathways involved in the differential expression genes.Results 1.In the survival analysis results,three regulatory factors(RBM15,METTL3,and LRPPRC)simultaneously showed the lowest P-value(P <0.1)and the highest hazard ratio(HR =1.99).Cox multivariate regression analysis of all variables also showed that the transfer(P <0.001,HR =4.038)significantly affected the overall survival rate of patients with osteosarcoma;2.Among the 21m6 A regulatory factors,methyltransferase RBM15 expression was significantly up-regulated in metastatic osteosarcoma samples compared with non-metastatic osteosarcoma samples;3.The Oncomine database showed that RBM15 displays an abnormally high copy number in four osteosarcoma subtypes(chondroblastic osteosarcoma,telangiectatic osteosarcoma,fibroblastic osteosarcoma,and osteoblastic osteosarcoma),further indicating the critical role of methyltransferase RBM15 in osteosarcoma.We also found that RBM15 was highly expressed in a variety of tumors,and the expression level of RBM15 was correlated with disease-specific survival in a variety of tumors.4.An evaluation of the relationship between RBM15 and 33 tumor groups from the TCGA database suggests that RBM15 may also play an important role in the development of other types of cancers and show specific associations with patient survival.5.TIMER database analysis showed that RBM15 expression was negatively correlated with CD4+ T cells,macrophages,and dendritic cells(P <0.05)but not significantly correlated with the other three immune infiltrating cells(B cells,CD8+ T cells,neutrophils).2.The quantitative Real-time PCR analysis showed that RBM15 m RNA expression was significantly increased in metastatic osteosarcoma cell lines(MNNG and 143B)compared with non-metastatic osteosarcoma cell lines(HOS and MG63).In addition,RBM15 m RNA expression was significantly increased in metastatic tumor tissue compared with primary tumor tissue.2.Western Blot analysis showed that RBM15 protein expression was significantly increased in metastatic osteosarcoma cell lines(MNNG and 143B)compared with nonmetastatic osteosarcoma cell lines(HOS and MG63).Also,RBM15 protein expression was significantly increased in metastatic tumors compared with primary tumors.3.A strongly positive RBM15 was observed in lung metastatic tumor tissue sections compared with the primary tissue sections,evidenced by Immunohistochemical staining.3.The si RNA transfection significantly inhibited RBM15 expression in metastatic osteosarcoma cell lines.2.CCK8 and colony formation experiments showed that down-regulation of RBM15 could significantly inhibit the cell proliferation in osteosarcoma cell lines(MNNG and 143B).3.Transwell and scratch experiments showed that RBM15 knockdown could significantly inhibit the cell invasion and metastasis in osteosarcoma cell lines(MNNG and 143B).4.The CCK8 and colony formation experiments showed that inhibition of RBM15 could significantly inhibit the cell proliferation in the MNNG cell line.However,overexpression of RBM15 could significantly promote cell proliferation in the HOS cell line.2.Transwell and scratch experiments showed that inhibition of RBM15 significantly repressed the cell migration and invasion abilities in MNNG cell lines.At the same time,overexpression of RBM15 significantly promoted the migration and invasion abilities of HOS cell lines.3.Compared with the control model,down-regulation of RBM15 reduced the volume of subcutaneous tumor formation in nude mice.4.Down-regulation of RBM15 can inhibit the formation of lung metastases in nude mice after tail vein injection.5.The differential expression genes were obtained in the RBM15 knocked down or overexpressed cells by Next-generation sequencing analysis."GO" analysis showed that differential expression genes were enriched in cell adhesion,apoptosis,and protein binding after RBM15 deletion.Meanwhile,the differential expression genes were mainly enriched in cell adhesion,apoptosis,and protein binding after RBM15 overexpression.For the "KEGG" analysis,after inhibition of RBM15,the differential expression genes were mainly enriched in NFκB and NLRs signaling pathways.While after overexpression of RBM15,the differential expression genes were mainly distributed in PI3K/AKT and NLRs signaling pathways.Of note,multiple pathways have been revealed to regulate tumor metastasis and progression.Conclusion 1.Abnormal expression of m6 A RNA methylation regulator RBM15 may play an important role in metastasis and prognosis of osteosarcoma;2.Methyltransferase RBM15 is overexpressed in metastatic osteosarcoma tissues and metastatic osteosarcoma cell lines,which may be related to metastasis;3.Loss of RBM15 can inhibit the proliferation,migration,and invasion of osteosarcoma cells;4.Methyltransferase RBM15 influences the proliferation,migration,and invasion of osteosarcoma cell lines may by regulating NLRs,NFκB,and PI3K/AKT signaling pathways,thereby affecting the distant metastasis of osteosarcoma.