The Mechanism Of HPV18E6/E7 Regulating METTL3 Mediated M~6A RNA Modification In Cervical Cancer Cells

【Objective】N6-methyladenine(m~6A)is the most common modification in advanced biological RNA,and it is also a research hotspot in the field of RNA.The modification process is dynamic and reversible,which is mediated by three elements:“Writer”,“Eraser”and“Reader”.It participates in many biological processes including RNA splicing,nuclear transport,RNA stability and translation.Human papillomavirus(HPV)is a circular double-stranded DNA virus,in which types 16 and18 are two common high-risk carcinogenic types,which usually participate in the regulation of multiple biological processes,and ultimately lead to the occurrence of cervical cancer.METTL3,as a methyltransferase,catalyzes the m~6A modification of RNA in vivo and in vitro,and participates in the regulation of many carcinogenesis processes,including hepatocellular carcinoma,but the relationship between m~6A modification and human papillomavirus E6/E7 protein in cervical cancer cells is not clear.In this paper,the regulatory mechanism between HPV18E6/E7 and METTL3 in cervical cancer cells was studied through a series of experiments,and the m RNA,that specifically interacted with METTL3 was determined by high-throughput sequencing of RNA binding protein immuno-(RIP)assay.Finally,the mechanism of METTL3-mediated m~6ARNA modification in the development of cervical cancer was elucidated.【Methods】First of all,the effect of HPV18E6/E7 on METTL3 m RNA and protein expression in cervical cancer cells C33A and He La was detected by dual luciferase reporter system,RT-q PCR and Western Blotting experiments.Bioinformatics software was used to predict the possible transcription factors bound to the METTL3promoter,and The dual luciferase reporter system and the Ch IP experiment were used to detect the regulatory effect of the METTL3 promoter.then RT-q PCR and Western Blotting experiments to detect the regulatory relationship between HPV18E6/E7,NR3C1 and METTL3 on the m RNA and protein levels.Then we used Me-RIP assay to detect the regulation of METTL3 on HPV18E6/E7m~6A methylation level,RT-q PCR and Western Blotting experiments to verify the effect of METTL3 on the transcription and translation of HPV18E6/E7.After that,the regulation of YTHDF1on HPV18E6/E7 m RNA and protein expression was verified by RIP-q PCR and Western Blotting experiments.Next,in order to further clarify the role of METTL3 in cervical cancer cells,we screened the genes interacting with METTL3 by high-throughput sequencing,and detected the effect of METTL3 on m~6A methylation level and m RNA by Me-RIP and RT-q PCR experiments.Finally,Western Blotting assay was used to detect the effect of HPV18E6/E7 on its protein expression.【Results】HPV18E6/E7 can upregulate the expression of METTL3 promoter,m RNA and protein.Cell transcription factor NR3C1 promotes its transcription and translation by binding to METTL3 promoter.In addition,NR3C1 mediates the upregulation effect of HPV18E6/E7 on METTL3.HPV18E6/E7 had a high level of m~6A methylation,its up-regulation depended on METTL3 enzyme activity center.In addition,METTL3 promotes the expression of HPV18E6/E7 m RNA,prolongs the half-life,and up-regulates its protein expression level.And YTHDF1 as a reader can interact with HPV18E6/E7 m RNA and promote its translation.Finally,the gene PER1,interacting with METTL3 was screened by RIP-seq high-throughput sequencing and Western Blotting experiments.METTL3 can regulate its m6A methylation modification to inhibit transcription.In addition,HPV18E6/E7 could inhibit the expression of PER1 protein.【Conclusions】The main purpose of this study is to explore the mechanism of HPV18E6/E7-regulated RNAm~6A methylation in cervical cancer cells.The results showed that HPV18E6/E7 protein up-regulated the expression of NR3C1,while NR3C1 bound to the promoter of METTL3 and up-regulated its expression.METTL3can increase the m~6A methylation level of HPV18E6/E7 m RNA,YTHDF1 can bind to HPV18E6/E7 RNA,and promote its translation,thus forming a positive feedback pathway between them.Further experimental show that METTL3 can down-regulate the m~6A methylation of tumor suppressor gene PER1,and finally play a role in promoting the occurrence and development of cervical cancer.