HPV E6原癌蛋白下调长链非编码RNA TUG1促进宫颈癌细胞增殖和迁移机制研究

The local persistent infection of human high risk HPV is a major factor that contributes to cervical cancer development.Moreover,HPV E6 DNA will be integrated into the host cell's DNA,and its' protein product would inactivate P53.P53 could promote LncRNA TUG1 transcription,and TUG1 may regulate target genes expression by binding PRC2.In our previous study,we found that TUG1 was downregulated in cervical cancer tissues compared with normal tissues,and knockdown of TUG1 expression could promote cells proliferation and invasion.Furthermore,knockdown of EZH2 expression could increase HOXB7 protein level.Therefore,we make a hypothesis that inactivation of P53 caused by HPV E6 oncoprotein leads to decreased TUG1 expression, which contributes to cervical cancer cells proliferation and invasion by regulating HOXB7 expression via binding PRC2. We will documented this mechanism by using RIP and CHIP et al methods,and our work will further our understanding about the molecular mechanisms of cervical cancer development and provide theoretical basis for early diagnosis and intervention of cervical cancer.

宫颈癌的主要致病因素为高致病基因型HPV局部持续感染，HPV DNA将以E6为主的基因片段整合到宿主细胞的DNA中，其基因产物E6原癌蛋白使细胞抑癌基因P53失活。P53能促进LncRNA TUG1的转录，TUG1可绑定PRC2调控相关基因表达。课题组前期在组织水平、细胞水平研究结果显示：宫颈癌细胞中TUG1表达下调，干扰TUG1后细胞的增殖和迁移能力增强，干扰PRC2复合物关键因子EZH2后HOXB7基因表达上调。因此提出假设：HPV E6原癌蛋白使P53失活可下调TUG1表达，TUG1可绑定PRC2进一步下调HOXB7基因，最终促进宫颈癌细胞增殖和迁移。本课题将通过大样本组织标本验证并在细胞和活体动物水平利用RIP、CHIP等技术阐述上述假设。本研究将进一步丰富宫颈癌的发病机制，为宫颈癌的早期诊断和干预提供理论依据。

长链非编码RNA（long noncoding RNA，LncRNA）近年来成为肿瘤相关研究的热点。一种名为尿路上皮癌相关抗原1（urothelial cancer associated 1，UCA1）的LncRNA在促进肿瘤细胞生长增殖、侵袭转移、化疗药物耐用等多种恶性表型方面发挥重要的功能。然而目前对UCA1的研究仍处于起步阶段，背后对的具体分子机制尚不十分明确。本项目通过荧光定量PCR（Real-time Quantitative polymerase chain reaction，RT-qPCR）检测宫颈癌、宫颈上皮内瘤变、对照组组织及脱落细胞，发现UCA1在宫颈癌及宫颈上皮内瘤变中高表达。通过双荧光素酶报告基因实验鉴定UCA1核心启动子区域为“-2112~-2090碱基”及“-2069~-2046碱基”，通过染色质免疫共沉淀实验（chromatin immunoprecipitation assay, CHIP）证明核心转录因子为CEBPA及FOXL1、FOXL4、FOXL6。UCA1A高表达稳转细胞系中UCA1A12倍高表达，两株细胞通过RNA-Seq测序及生物信息学分析，结果显示UCA1a参与多方面的生物学功能进程。经RNA结合蛋白免疫沉淀（RNA Binding Protein Immunoprecipitation Assay，RIP）、质谱分析发现能与UCA1a的相互作用蛋白Pyruvate、kinase、PKM2等，进一步实验显示PKM2能与UCA1a的正义链相互作用，PKM2的免疫沉淀产物中含有UCA1a，UCA1a中的400-800碱基区间片段能与PKM2结合，PKM2的A1、B、A2结构域能与UCA1a结合。通过Western Blot 检测PKM2在核内表达，发现UCA1a能够增加PKM2的蛋白稳定性和促进PKM2入核，从而增加PKM2的活性。细胞功能学实验证实，UCA1a依赖PKM2促进细胞的增殖及迁移。