HPV整合至8q24区域长链非编码RNA CCAT1位点的检测及其在宫颈癌中作用机理的研究

HPV integration can retain promoting sequence and oncogenes, and induce genomic instability. Long non-coding RNA CCAT1 can promote proliferation and regulate oncogene MYC as an enhancer. The previous work of this project showed by high-throughput sequencing that, HPV integrated in the human genome 8q24 locus CCAT1 with high-frequency. Copy number of 8q24 locus was increased significantly in cervical cancer. We speculate that: HPV can integrate in the 8q24 locus, regulate CCAT1 and relating signaling pathway, and promote the occurrence and development of cervical cancer. The project is designed to explore the role of CCAT1 in cervical cancer and molecular mechanisms by over-expression, knock down, pull down and RIP techniques. To explore cell phenotype and regulation of CCAT1 in the 8q24 region by exogenous promoting sequence, by constructing genome editing TALEN to knock in and knock out HPV upstream regulatory sequences. To explore the value of HPV integration at 8q24 locus as cervical cancer biomarkers. This research project is to make a meaningful exploration on the role of HPV integration in the development of cervical cancer.

HPV整合可以保留病毒启动序列和癌基因，并诱导整合位点附近的不稳定性。近年发现长链非编码RNA CCAT1具有促进增殖作用，并且作为增强子顺式调控癌基因MYC的表达。本项目前期工作通过高通量测序等技术显示，HPV高频整合在人基因组8q24区域CCAT1位点，宫颈癌中8q24位点的拷贝数水平升高。在此基础上我们推测：HPV整合在8q24区域后调控该位点的CCAT1和与其相关信号通路，进而促进宫颈癌的发生发展。本项目拟采用过表达、敲降、pull down、RIP等技术探索CCAT1在宫颈癌中的作用和分子机制。构建基因组编辑作用的TALEN敲入和敲除HPV，探索HPV病毒整合至8q24区域对细胞性状的作用和对CCAT1及其相关信号通路的调控作用。探索以HPV整合在8q24区域CCAT1位点作为CIN向宫颈癌进展的生物学标记物。本项目研究为HPV整合在宫颈癌发生中的作用机制作出有意义的探索。

本项目在宫颈癌病人的新鲜组织标本中证实，HPV在8q24整合位点处的CCAT1在宫颈癌组织里的表达水平普遍高于其癌旁组织。细胞实验证实CCAT1具有促进宫颈癌细胞增殖和侵袭恶性表型的作用。细胞和体内实验证实CCAT1调控miR-181a-5p/MMP14信号通路发挥作用，并促进裸鼠皮下瘤生长。从临床转化角度，合成了基于PBAE材料和CRISPR质粒的纳米颗粒，用于靶向敲除HPV癌蛋白表达, 为合成CCAT1纳米靶向药物奠定基础。本项目阐明了长链非编码RNA CCAT1在宫颈癌发生发展中的重要作用，相关的分子机制，并建立了基于PBAE的临床转化应用基础，CCAT1作为宫颈癌发病的分子标记物和治疗靶点值得进一步研究。