Construction Of Transcriptome Map Of M6A-related Genes And Mechanism Of Key Gene DRG1 In Osteosarcoma

Objective: Osteosarcoma(OS)is a malignant bone tumor commonly seen in children and adolescents,and its incidence has been increasing in recent years.The development of osteosarcoma is a multi-mechanism,multi-regulated process involving changes in genomics and epigenetics.N6-methyladenosine(m6A)is a research hotspot on the epigenetic modification of RNA molecules,which can affect RNA transcription,splicing,structural changes,localization,nuclear transport,translation and degradation.Increasing evidence suggests that changes in m6 A levels and dysregulation of m6 A are related to the occurrence,development,and prognosis of OS.At present,the aberrant methylation targets associated with osteosarcoma are still understudied,and the specific mechanisms are different.Combining current bioinformatics provides good conditions for finding new targets,therefore,we designed and carried out this study to use bioinformatics analysis to find more sensitive and specific abnormal nails that are significantly associated with osteosarcoma.Synthesis targets and biomarkers,while exploring the expression and specific regulatory mechanism of m6 A RNA modification in osteosarcoma,looking for key regulatory genes that cause abnormal biological behavior of osteosarcoma,and analyzing the role of this regulatory network in the occurrence and development of osteosarcoma And how to play the role mechanism in prognosis,to provide new potential biological targets for the diagnosis and treatment of osteosarcoma.Methods:(1)Obtained by bioinformatics analysis whether there are m6 A differential genes between normal human osteoblasts and osteosarcoma cell lines and between metastatic osteosarcoma and non-metastatic osteosarcoma tissues;if so,screen out the differentially methylated sites point expressed genes.Multivariate COX regression analysis was used to analyze the correlation between m6 A differential genes and the prognosis of patients with osteosarcoma,and to analyze the risk factors for poor prognosis of osteosarcoma,and to preliminarily screen the factors that play a key role in regulating m6 A modification in osteosarcoma.(2)Combined with the current research upsurge of long non-coding RNAs,four differential genes closely related to the prognosis of osteosarcoma patients were screened for further research through bioinformatics,and a co-expression network map of the visualized m6 A gene and related lnc RNAs was constructed.Univariate COX regression analysis and Lasso regression analysis were used to construct osteosarcoma-related prognostic models based on multiple databases,and to further clarify the important role of the screened differential gene m6A-related lnc RNAs in the prognosis and immune microenvironment of OS patients.provide a theoretical basis.(3)Based on the results of previous studies,METTL3 was selected as a candidate gene for exploring the disorder of RNA m6 A modification in osteosarcoma.The prediction was evaluated by TCGA and GEO database,combined with Pearson correlation analysis to explore the correlation between DRG1 and m6 A gene.The METTL3-silencing osteosarcoma cell line was constructed by transfection of small interfering RNA(si RNA)technology.RT-q PCR,Western blotting(WB)and m RNA stability experiments were used to explore the correlation between METTL3 and DRG1 expression.The downstream regulatory target genes of METTL3 in osteosarcoma were verified.(4)In osteosarcoma cell lines MG63 and 143 B,after transfection of si RNA to silence the expression level of DRG1,CCK-8 assay,Colony plate cloning assay,Trans-well assay,wound healing scratch assay,flow cytometry and other methods were used.To explore the effect of abnormal expression of DRG1 on the biological function of osteosarcoma cells,in order to further clarify the biological role of DRG1 in the occurrence and development of osteosarcoma.Results:(1)Bioinformatics analysis showed that m6 A RNA modification was highly expressed in osteosarcoma cell lines.Compared with the corresponding adjacent tissue,the expression level of m6 A RNA modification in osteosarcoma tissue was significantly higher than that in adjacent tissue.(2)Bioinformatics screened out 4 major m6 A regulated genes with differences in osteosarcoma.Multivariate COX regression analysis showed that METTL3,LRPPRC,YTHDF2 and FTO were closely related to the prognosis of patients with osteosarcoma.Risk factors for poor prognosis.By analyzing the lnc RNA expression of four differential genes including METTL3,LRPPRC,YTHDF2 and FTO and m6A-related lnc RNA,the co-expression network map of m6 A gene and related lnc RNA was constructed to visualize the m6 A gene.Univariate COX regression analysis and Lasso regression analysis were used to construct an osteosarcoma-related prognostic model based on multiple databases.It was found that the high expression of m6 A methyltransferase METTL3 may be an important reason for the up-regulation of m6 A RNA modification in osteosarcoma.(3)The predictions were evaluated by TCGA and GEO databases,and combined with Pearson correlation analysis,it was found that the expression of METTL3 and DRG1 were the most correlated.In osteosarcoma cell lines MG63 and 143B,silencing of METTL3 resulted in decreased DRG1 m RNA and protein levels and impaired DRG1 m RNA stability to varying degrees.(4)Knockdown of DRG1 can significantly promote the G1 phase arrest and inhibit the proliferation,migration and invasion of osteosarcoma cells.The results suggest that DRG1 may play an important tumor-promoting role in the occurrence and development of osteosarcoma,and METTL3 is regulated in an m6A-dependent manner.The expression of DRG1 promotes the occurrence and development of osteosarcoma.Conclusion:(1)The m6 A RNA modification was significantly up-regulated in osteosarcoma tissues and cell lines,and the high expression of m6 A methyltransferase METTL3 may be an important reason for the up-regulation of m6 A RNA modification in osteosarcoma.(2)The candidate gene METTL3 with disordered RNA m6 A modification in osteosarcoma has a certain correlation with the downstream target gene DRG1.Silencing of METTL3 resulted in decreased DRG1 m RNA and protein levels,and impaired DRG1 m RNA stability to varying degrees.(3)Knockdown of DRG1 can significantly promote the G1 phase arrest and inhibit the proliferation,migration and invasion of osteosarcoma cells.The results suggest that DRG1 may play an important tumor-promoting role in the occurrence and development of osteosarcoma,and METTL3 is regulated in an m6A-dependent manner.The expression of DRG1 promotes the occurrence and development of osteosarcoma.