【作者】 陈曦；

【导师】 汤华；

【作者基本信息】 天津医科大学 ， 病原生物学， 2020， 硕士

【摘要】 【目的】N~6-甲基腺嘌呤(N6-methyladenosine,m~6A)是发生在高等生物RNA中最为普遍的修饰,也是当前RNA领域研究的热点。其修饰过程动态可逆,是由“编码器(Writer)”、“消码器(Eraser)”和“读码器(Reader)”三个元件共同介导,参与了包括RNA剪切、核转运、RNA稳定性和翻译在内的多个生物学过程。人乳头瘤病毒(Human Papillomavirus,HPV)是一种环状双链DNA病毒,其中16和18型是两个常见的高危致癌型别,通常参与调控多个生物学过程,最终导致宫颈癌的发生。甲基化转移酶样蛋白3(methyltransferase like protein 3,METTL3)作为甲基转移酶在体内外催化RNA发生m~6A甲基化修饰,参与包括肝癌在内的多个癌发生过程的调节,但其与宫颈癌细胞中人乳头瘤病毒E6/E7蛋白之间的关联尚不清楚。本文主要是通过一系列实验研究宫颈癌细胞中HPV18E6/E7与METTL3之间的调控机制,通过RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)实验高通量测序确定与METTL3特异性相互作用的m RNA,最终阐明METTL3介导的m~6A RNA修饰在宫颈癌发生发展过程中的作用机制。【方法】首先,通过双荧光素酶报告系统、RT-q PCR和Western Blotting实验检测了宫颈癌细胞C33A和He La中HPV18E6/E7对METTL3 m RNA和蛋白表达的影响。生物信息学软件预测了METTL3启动子上可能结合的转录因子,并通过双荧光素酶报告系统和染色质免疫共沉淀(Ch IP)实验检测NR3C1对METTL3启动子的调控作用,随后RT-q PCR和Western Blotting实验检测HPV18E6/E7、NR3C1及METTL3三者在m RNA和蛋白表达水平的调节关系。接着我们通过甲基化RNA免疫沉淀(Me-RIP)实验检测METTL3对HPV18E6/E7m~6A甲基化水平的调节,RT-q PCR和Western Blotting实验验证METTL3对HPV18E6/E7转录和翻译水平的影响。之后,通过RIP-q PCR和Western Blotting实验验证YTHDF1对HPV18E6/E7 m RNA作用和蛋白表达的调节。接下来,为进一步明确METTL3在宫颈癌细胞中的作用,我们通过RIP-seq高通量测序筛选了与METTL3相互作用的基因,并通过Me-RIP和RT-q PCR实验检测METTL3对其m~6A甲基化水平和m RNA表达的影响,最后Western Blotting实验检测HPV18E6/E7对其蛋白表达的影响。【结果】HPV18E6/E7可上调METTL3启动子、m RNA和蛋白表达水平,细胞转录因子NR3C1通过结合到METTL3启动子上促进其转录和翻译,并且NR3C1介导了HPV18E6/E7对METTL3的上调作用。HPV18E6/E7本身具有较高的m~6A甲基化水平,并且METTL3对它们m~6A甲基化水平的上调作用依赖酶活性中心。此外,METTL3能上调HPV18E6/E7 m RNA的表达水平,延长半衰期,并促进其蛋白表达。YTHDF1作为reader可与HPV18E6/E7的m RNA相互作用并促进其翻译。RIP-seq高通量测序和Western Blotting实验筛选出与METTL3相互作用的基因PER1,METTL3可调节其m RNA m~6A甲基化修饰抑制转录。除此之外,HPV18E6/E7抑制了PER1的蛋白表达。【结论】本实验主要探究HPV18E6/E7调节的RNA m~6A甲基化在宫颈癌细胞中作用机制。结果表明HPV18E6/E7蛋白上调NR3C1的表达,而NR3C1结合METTL3的启动子并上调其表达。METTL3可增加HPV18E6/E7 m RNA的m~6A甲基化水平,YTHDF1同样能结合HPV18E6/E7的RNA,并促进其翻译,因而它们之间形成一个正反馈通路。进一步实验结果阐明METTL3能够调节抑癌基因PER1的m~6A甲基化修饰,并最终发挥共同促进宫颈癌发生发展的作用。更多还原

【Abstract】 【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.更多还原