PL2L60在HPV转化细胞中表达重启及其调控子宫颈癌干细胞形成的分子机制

Previous studies have demonstrated that PL2L60 played an important role in gene regulation, which normally expressed in germline stem cell. Moreover, in carcinomas PL2L60 specially expressed in pre-cancer stem cells (pCSC) and cancer stem cells (CSC), which most likely attribute to tumorigenesis. It is widely accepted that high-risk human papillomavirus (HR-HPV) infects cervical epithelial reserved cells (CERC) through its receptor of Integrinα6β4, leading to malignant transformation as the DNA of HR-HPV integrated into host DNA. In cervical cancer, the theory of cancer stem cell indicated that the pCSC and CSC were derived from HR-HPV transformed CERC. Our latest studies showed that PL2L60 enhanced the capability of tumorigenesis of HaCaT cells transfected by PL2L60 via activating Stat3/Bcl-XL signaling pathway after transplanted in nude mice. On the contrary, the tumorigenesis capability was significantly suppressed in SiHa cell as PL2L60 was silenced via shRNA. Therefore, it deserved further studies how HR-HPV restored the expression of PL2L60 in the CERC and what role of PL2L60 acted in the genesis of cervical cancer stem cells. In this study, we upregulated and silenced the E6, E7, and PL2L60 expression in the cell lines of HaCaT and W12E/G, CERC, and CSC either by transfection or by shRNA, which,to some extend, well mimiced the pathological changes in the process of tumorigenesis in vitro. Consequently, we examined the activity of PL2L60 in the gene modulation, further clarified the mechanisms of PL2L60 re-expression and its role in tumorigenesis of cervical carcinoma. The discovery of this study will definitely contribute to the enhancement of the pathogenesis of cervical cancer, and explore new targets for blocking the progression of cervix carcinogenesis induced by HR-HPV.

研究证实，PL2L60具有强大基因调控作用，在肿瘤中特异表达于癌前干细胞（pCSC）和肿瘤干细胞（CSC），与肿瘤发生密切相关；子宫颈癌的发病是由高危型人乳头瘤病毒（HR-HPV）通过Integrinα6β4感染子宫颈上皮储备细胞（CERC），引发CERC向pCSC和CSC转化所致。课题组前期发现，HaCaT细胞转染PL2L60通过激活Stat3/Bcl-XL途径而恶性转化，而沉默PL2L60可显著降低SiHa细胞致瘤性。因而，作为早期分子事件PL2L60在CERC恶性转化中的作用凸显关键。本课题拟选择CERC、正常角化细胞株HaCaT、癌前病变来源细胞株W12E/G和宫颈癌CSC为研究对象，体外模拟宫颈癌病变进程，通过上调和沉默PL2L60表达，探讨HR-HPV感染重启PL2L60表达及其调控CERC向肿瘤干细胞转化的分子机制，从而完善子宫颈癌发病机制，为阻断子宫颈病变进展寻找特异靶点。

高危型人乳头瘤病毒（high risk human papillomavirus, HR-HPV）持续感染，E6、E7表达使宫颈上皮发生转化及永生化，形成宫颈癌干细胞，但是具体机制尚不清楚。我们前期研究发现，仅在胚胎时期和睾丸中表达的干细胞分子Piwil2在宫颈上皮内瘤变（cervical intraepithelial neoplasia, CIN）Ⅱ、CINⅢ及宫颈癌干细胞中重现表达，实验证实为PL2L60（Piwil2 like 60），其与肿瘤发生的关系尚待明确。本项目深入探讨HR-HPV转化细胞重启PL2L60表达的分子机制，以及PL2L60在宫颈癌肿瘤形成细胞（tumor initiating cells, TICs）形成中的作用机制。我们的实验结果证实，PL2L60仅在HPV阳性宫颈癌细胞株表达，而HPV阴性的C33A细胞株则未见表达；抑制宫颈癌细胞PL2L60表达在体内外均显示良好的抗肿瘤作用；在HaCaT细胞中过表达PL2L60，则显著提高了细胞增殖、侵袭能力，促进EMT转化，使不具备成瘤能力的HaCaT细胞获得了很强的致瘤性；高通量表达谱芯片GSEA分析发现，PL2L60能显著上调ESC相关核心转录因子c-myc、nanog、oct4、sox2、klf4及其靶基因的表达丰度，上调ESC不同时相、EMT及肿瘤进展相关基因的表达。慢病毒介导HR-HPV E6E7过表达，结果显示E6E7能重启PL2L60表达，GSEA分析显示与PL2L60分子相似的干细胞相关基因表达谱的调控作用。进一步结果证实，PL2L60通过与H3K9ac与c-myc、nanog、oct4、sox2、klf4基因promoter区域结合，上调转录因子及其靶基因的表达，从而使体细胞重编程形成TICs，导致宫颈癌发生。本项目首次阐明了宫颈癌干细胞形成的分子机制，进一步完善了HR-HPV E6E7致宫颈癌的发病机制，为宫颈癌前病变及癌的治疗提供了新的干预靶点。