TET1介导的自噬对宫颈癌细胞生物学行为的影响及机制研究

Abnormal methylation of autophagy genes is closely associated with tumorigenesis. TET1 is one of the newly discovered DNA methylation enzyme genes. It was found that the expression level of TET1 was down regulated in solid tumors such as colon cancer, prostate cancer and breast cancer etc, and probably plays a role of tumor suppressor genes. We also found that the expression level of TET1 was negatively correlated with pathologic stage of cervical cancer clinical. Furthermore, the level of autophagy related genes LC3B and Beclin 1 were down regulated along with the decrease of TET1 in cervical cancer cells. These results suggested that there were some correlations between TET1 and autophagy in cervical cancer. Thus we speculate that TET1 may regulate the biological behavior of cervical cancer cells by adjusting the methylation level of autophagy related genes. Therefore, this project is mainly designed to verify our speculation. The established cervical cancer cell lines HeLa and Caski will be used for the study. Transmission electron microscopy, immunohistochemistry, Western blot, Dot blot, ChIP, hMeDIP, CRISPR/Cas9 targeting and other related technologys, as well as cells and animal experimental model will be used to detect the effect and mechanism of TET1-mediated autophagy on cervical cancer biology behavior. Through this study, we hope to find a new method and target for the clinical treatment of cervical cancers.

自噬对维持细胞内环境稳定有重要意义，异常自噬与宫颈癌发生密切相关。自噬基因甲基化异常是自噬调节肿瘤的重要方式之一。TET1是新发现的一个DNA去甲基化酶基因，研究发现其在结肠癌、乳腺癌等实体癌中表达下调，可能起抑癌基因作用。课题组前期研究发现宫颈癌组织中TET1的表达与宫颈癌病理分级负相关，而且宫颈癌细胞中随着TET1表达水平的降低自噬相关基因表达上调，这提示宫颈癌中TET1与自噬之间存在一定的关联。由此我们推测：TET1可能通过调节自噬相关基因的甲基化水平进而参与对宫颈癌细胞生物学行为的调控。本研究将以宫颈癌HeLa、Caski细胞为研究对象，采用透射电镜、免疫组织化学、Western blot、Dot-blot、ChIP、hMeDIP及CRISPR/Cas9打靶等相关技术，结合细胞和动物实验模型，明确TET1在调节宫颈癌细胞自噬水平并影响其生物学行为中的相关机制。

自噬对维持细胞内环境稳定有重要意义，异常自噬与宫颈癌发生密切相关。自噬基因甲基化异常是自噬调节肿瘤的重要方式之一。TET1是新发现的一个DNA去甲基化酶基因，研究发现其在结肠癌、乳腺癌等实体癌中表达下调，可能起抑癌基因作用。为了研究TET1是否通过调节自噬参与宫颈癌生物学行为的调节，本研究通过敲出、过表达及转录组测序等方法，观察TET1对宫颈癌细胞自噬及增殖、侵袭、迁移等生物学行为的影响，阐明TET1抑制宫颈癌恶性行为的机制。首先我们通过分析TCGA、GTEx数据库，并收集宫颈癌组织标本进行免疫组化及Western blot检测，发现宫颈癌组织中TET1 表达水平低于正常对照组。且宫颈癌组织中5mC丰度与宫颈癌程度负相关，而5hmC丰度有宫颈癌分化程度正相关。通过对宫颈癌细Siha及HeLa细胞TET1基因进行敲出和过表达，发现TET1敲出能促进宫颈癌细胞的增殖、侵袭、迁移、单克隆形成及体内成瘤能力，而TET1过表达则能抑制宫颈癌的以上恶性行为学行为。说明TET1对宫颈癌具有抑制作用。此外我们还发现TET1基因敲出可以降低宫颈癌细胞自噬水平，而过表达则能促进自噬。说明TET1对宫颈癌细胞自噬具有正向调控作用。对TET1基因敲出及野生型细胞进行转录组测序，发现TET1调控宫颈癌细胞的信号通路包括自噬、凋亡、mTOR、PI3K/Akt等。筛选出13个差异表达自噬相关基因，进一步分析宫颈癌中这些差异自噬基因与TET1的相关性，发现与TET1正相关的自噬基因有ATG4B、TSC1及PIK3C3。进一步对宫颈癌中TET1正向调控的这3个自噬相关基因甲基化进行分析，发现PIK3C3及ATG4B均存在多个甲基化区域与宫颈癌患者生存密切相关。提示TET1调控宫颈癌自噬可能是通过调节ATG4B及PIK3C3基因的甲基化来实现的。总的来说，本研究发现：①宫颈癌TET1表达明显低于正常宫颈组织，且与宫颈癌分化程度呈正相关；②TET1在宫颈癌中起抑癌基因作用；③ TET1对宫颈癌细胞自噬起正向调控作用；④TET1调控宫颈癌细胞恶性生物学行为可能是通过调节ATG4B及PIK3C3基因的甲基化进而调控自噬来实现的。