锌指蛋白ZNF677的表观遗传学失活在甲状腺癌发生及发展中的作用

Thyroid cancer is the commonest endocrine malignancy with a rapidly rising incidence in many regions of the world. However, the pathogenesis of this disease is largely unknown. Like other human cancers, initiation and progression of thyroid cancer involves multiple genetic ad epigenetic alterations, ultimately causing gain-of-function in oncogenes and loss-of-function in tumor suppressor genes. Zinc-finger proteins are the the largest family of transcription factors with zinc finger motif, which play a key role in cell differentiation and embryogenesis. Most of them are transcription repressors. However, the repression is commonly realized via creation of an inactive chromatin domain rather than via direct influence on preinitiation complex assembly, such histone deacetylation, histone methylation, recruitment of HP1 protein, and formation of a sufficiency stable repressed domain (heterochromatin). The previous studies show that several Zinc-finger proteins are aberrantly expressed in human cancers, contributing to human carcinogenesis. However,molecular mechanism remains poorly understood, particularly in thyroid cancer. Of note, our preliminary data showed that ZNF677 gene was down-expressed in primary thyroid tumors and thyroid cancer cells compared with normal thyroid tissues. Moreover, this gene was frequently hypermethylated in a cohort of thyroid cancers. Demethylation agent 5-Aza-2'-dC and histone deacetylase inhibitor SAHA significantly induced re-expression of silenced ZNF677 gene in thyroid cancer cells. Importantly, restoring ZNF677 expression in thyroid cancer cells dramatically suppressed cell proliferation, colony formation, invasion and migration, and induced cell cycle arrest and apoptosis, suggesting that ZNF677 may be a novel tumor suppressor in thyroid cancer. The aim of this project is to further explore biological role and molecular mechanisms of ZNF677 in thyroid carcinogenesis, and identify its downstream target genes using ChIP-Seq and gene expression microarray approaches.

甲状腺癌如今已成为发病率最高的内分泌系统肿瘤，其发生涉及诸多的遗传及表观遗传学改变，最终导致癌基因的过度表达及抑癌基因的失活。锌指蛋白是一类具有手指状结构域的转录因子，且多为转录抑制因子，其转录抑制大多通过组蛋白去乙酰化、组蛋白甲基化、招募HP1蛋白及形成兼性异染色质来实现。这类转录因子不但在细胞分化和胚胎发育等方面起到重要的调节作用，而且与恶性肿瘤的发生密切相关。我们前期结果发现锌指蛋白ZNF677在甲状腺癌组织及细胞中表达下调，且其启动子区发生高频率的DNA甲基化，DNA去甲基化试剂和组蛋白去乙酰化酶抑制剂能有效恢复其表达。恢复该基因的表达能显著抑制肿瘤细胞增殖、克隆形成、侵袭及迁移能力，并诱导细胞发生周期阻滞及凋亡，提示ZNF677可能是一个新的甲状腺癌抑制因子。本项目在此基础上，进一步明确ZNF677的生物学功能，鉴定其基因调控网络，进而阐明其参与甲状腺癌发生与发展的分子机制。

甲状腺癌是内分泌系统最常见的恶性肿瘤，且近年来发病率逐年上升。随着对甲状腺癌发病机制的深入研究，PI3K/Akt及MAPK/Erk这两条信号通路在甲状腺癌发生和发展中的核心地位已突显无疑。这两条信号通路的异常激活不仅参与甲状腺癌生长和转移，并且是分化型甲状腺癌向低分化或未分化型甲状腺癌进展的主要动力，目前已经成为甲状腺癌治疗的主要分子靶点。遗传学及表观遗传学机制共同参与肿瘤发生和发展过程。DNA甲基化引起的表观遗传学改变是导致多种抑癌基因在肿瘤中失活的重要机制。ZNF677属于KRAB型锌指蛋白，锌指蛋白通过结合特定的DNA发挥转录因子作用。有研究发现锌指蛋白ZNF677在非小细胞肺癌中发挥抑癌作用，但ZNF677在恶性肿瘤中的抑癌机制并不明确，特别是在甲状腺癌中至今未见相关研究。我们在前期工作中发现，与正常甲状腺组织相比，ZNF677基因在甲状腺癌组织及细胞中表达显著下调，且该基因的启动子区发生高频率的DNA甲基化。ZNF677的低表达与甲状腺癌患者的不良预后显著相关。恢复该基因的表达能显著抑制肿瘤细胞的增殖、克隆形成、侵袭及迁移以及裸鼠成瘤能力，并且可以诱导肿瘤细胞发生周期阻滞及凋亡。反之，敲减ZNF677基因则促进甲状腺癌细胞的生长及侵袭能力，这些结果提示ZNF677可能是一个新的甲状腺癌抑制因子。进一步的研究证实，ZNF677通过抑制其下游的两个靶基因CDKN3和HSPB1的转录调控Akt Ser473的磷酸化，进而抑制甲状腺癌的进展。