The Mechanism Of METTL3-Mediated M~6A Modification Regulating The Translation Of Circ-TNFRSF19 To Promote Malignant Progression Of Glioma

Glioma is the most common primary malignant tumor of the central nervous system,among which Glioblastoma(GBM)is the most malignant,and the phenomenon of recurrence and chemoradiotherapy resistance is obvious.Despite substantial improvements in concomitant surgical modalities and adjuvant therapeutic measures,its prognosis is still far from satisfactory,and the average median survival time of GBM patients is only 14.6 months.Therefore,it is crucial to understand the molecular mechanisms underlying the origin and progression of GBM.Tumor initiation or progression is a complex process that often results from aberrant genetic or epigenetic changes.Epigenetic abnormalities mainly occur at the level of DNA,RNA,and histone modifications,and more than 100 types of post transcriptional modifications have been identified at the RNA level,with N~6-methyladenosine(m~6A)modification being one of the most common types,accounting for approximately 50%of total methylated ribonucleotides.As the most predominant form of m RNA modification in eukaryotic cells,m~6A modification is a reversible chemical process that is dynamically controlled by the balanced activities of methyltransferases and demethylases.Among them,methyltransferase like3(METTL3)as the first discovered and the most important methyltransferase,is involved in all stages of the RNA life cycle.It plays critical roles in pre-m RNA splicing,3’-end processing,nuclear export,translational regulation,m RNA decay and mi RNA processing.Thus,METTL3 is able to influence tumor formation by regulating m~6A modification in the m RNA of key oncogenes or tumor suppressors.Interestingly,besides m RNA,well-known non coding RNA-circular RNA(circRNA),a kind of covalently linked single stranded closed circular RNA molecule without 3’-end poly(A)tail and 5’-end cap,also has extensive m~6A modification sites,and circRNA is closely associated with glioma occurrence and development.Studies have shown that METTL3 can promote the formation of m~6A-circRNA while exhibiting a completely different pattern of m~6A modification from m RNA,but the mechanism by which METTL3 mediated m~6A modification regulates circRNAs and their downstream targets in the development and progression of glioma remains unknown.This study takes“m~6A modification regulates circRNA”and“RBP function of circRNA”as the starting point,and puts forward the scientific hypothesis that“m~6A modification mediated by methyltransferase METTL3 regulates circRNA/RBP pathway to promote the occurrence and development of glioma”.Research Objectives:Based on the m~6A sequencing and transcriptome sequencing of circRNA in the early stage of our research group,the function and mechanism hypothesis of“m~6A modification mediated by methyltransferase METTL3 regulates circRNA/RBP pathway to promote the occurrence and development of glioma”are going to be verified in combination with bioinformatics analysis,cell(in vitro)experiment,animal(in vivo)experiment and clinical level research.To explore the important role of m~6A modification in the biological origin and malignant progression of glioma,and enrich the interaction network of oncological regulation.It is expected to open a new perspective for the early diagnosis,pathological grading,targeted therapy and prognosis evaluation of glioma.At the same time,it also provides a certain theoretical and experimental basis for in-depth study of the key role of m~6A modification in regulating non coding RNA in glioma.The specific research contents are as follows:Research Methods:(1)Based on clinical GBM and normal non-tumor brain tissue samples,RNA methylation sequencing(m~6A-Seq)and RNA transcriptome sequencing(circRNA-Seq)were performed,and advanced bioinformatics analysis was performed for the sequencing results.The m~6A related interest circRNAs were immunoprecipitated,and the m~6A modification level and expression level of circRNA were verified by RT-q PCR,and the target circRNAs were screened.(2)Si RNA technology combined with RT-q PCR verification were used to complete RNA knockdown.Ed U experiment,Flow cytometry and Transwell experiment were used to test the functions of METTL3 and the selected target circRNAs in glioma cells.A stably transfected METTL3 knockdown cell model was constructed by lentivirus.The effect of METTL3 on glioma growth in vivo was studied by xenotransplantation in nude mice.(3)Target circRNA wild-type and mutant plasmids were constructed,Me RIP-q PCR(also m~6A-q PCR)and RT-q PCR combined with m~6A point mutation were used to explore the regulation of METTL3 on circRNA.Ed U experiment further verified the cancer-promoting effect of downstream target circRNA through Rescue.CHIRP combined with Mass Spectrometry was used to determine the target circRNA binding protein,and RIP was used to reverse confirm the binding effect.The circular properties of circRNA were verified by circRNA loop formation experiment.RT-q PCR,WB and Immunohistochemistry were used to detect the expression levels of targeted genes and proteins in nude mice.Research Results:(1)Compared with the NC group,there were 1370 novel m~6A peaks and 1322 missing m~6A peaks in circRNA from GBM.Among the sites associated with changes in m~6A peaks,1298 were up-regulated and 1905 were down-regulated.The level of m~6A modification was positively correlated with circRNA expression.Bioinformatics analysis predicted the biological functions and corresponding signaling pathways of m~6A modified circRNAs.In addition,three molecules of interest(TNFRSF19、DLGAP5 and NDC80)were identified through RT-q PCR validation combined with clinical data mining as preliminary candidate circRNAs to further investigate the functions and mechanisms by which m~6A modification mediates the development and progression of GBM.(2)Small interfering RNA transient and lentiviral stable knockdown RNA glioma cell model construction was successful.After the expression of METTL3 and circRNAs of interest(circ-TNFRSF19 and circ-NDC80)was down-regulated,the proliferation,invasion and migration of glioma cells were significantly reduced,and apoptosis was increased.In addition,nude mice xenograft tumors showed slower growth rate and reduced tumor mass and volume in vivo after METTL3 knockdown compared with controls.(3)There were extensive m~6A modification sites on circ-TNFRSF19 and its ring structure was confirmed by ring formation experiments.After METTL3 knockout,the methylation level and expression level of circ-TNFRSF19 were significantly down-regulated,and this regulatory effect was invalid after m~6A sequence point mutation.The results of recovery experiment showed that the inhibition of cell proliferation caused by METTL3 knockdown could be recovered by overexpression of circ-TNFRSF19.circ-TNFRSF19 can bind a variety of ribosomal proteins.Two possible translation products(The number of amino acids was148aa and 74aa,respectively)were predicted based on ORFfinder.Research Conclusions:(1)The findings of the present study provide the first map of m~6A modification of circRNA in human GBM and identify differentially expressed circRNA transcripts associated with hyper and hypo methylated modifications,revealing potential associations between aberrant m~6A modification and cancer-related gene expression and function.This will be important for further in-depth exploration of the regulation of circRNA expression and its function by RNA methylation modification.(2)The methyltransferase METTL3 significantly promotes glioma development in vivo and in vitro.The high m~6A modification levels and high expression levels of circ-TNFRSF19and circ-NDC80 obtained from targeted sequencing screening also enhance the proliferation,invasion and migration abilities of glioma cells,while inhibiting their apoptosis,and play important roles in promoting tumor malignant progression.(3)There are extensive m~6A modification sites on circ-TNFRSF19,and METTL3positively regulates the methylation level and transcription level of oncogenic circ-TNFRSF19 through an m~6A dependent manner and affect the tumor driving effect.Moreover,circ-TNFRSF19 can potentially translates several polypeptide products by binding to ribosomal proteins,and then plays a role in the malignant progression of glioma.In conclusion,this study highlights that METTL3-mediated m~6A modification plays a carcinogenic role in the development and progression of glioma by regulating circ-TNFRSF19 translation pathway.It not only provides experimental basis for in-depth understanding of the related mechanisms of circRNA influence on glioma under the regulation of m~6A modification,but also provides a new idea for targeted treatment of glioma.