CLDN4表达下调促进人喉癌细胞恶性表型的机制

In our previous work, We found that the expression of CLDN4 was down-regulated in laryngeal squamous cell carcinoma tissues and cell lines, DNA methylation is one of the reasons leading to the down-regulation of its expression, and Hep-2 cells wich dealed with 5-aza-dc grew slowly, anchorage-independent growth, invasive and migratory traits were also substantially decreased in cells with CLDN4 expression. These results suggest that CLDN4 may act as a cancer suppressor.In this study， ChIP、ChIP-reChIP、Nuclease accessibility assays was used to determine the mechanism of DNA methylation regulating the expression of CLDN4 1）by directly interfering with the binding of transcription factors 2）by the involvement of proteins that specifically recognize methylated CpGs, histone deacetylases and histone methyltransferase 3)DNA methylation could also repress gene transcription by mechanisms of chromatin condensation. And transfection, transepithelial electric resistance detection and western blot was used to determine the mechanism of CLDN4 in cancer development by 1) changed the tight junction fuction, 2)activation of sugnaling pathway, or3) effects on some proteins related with migration and invasive ability. We try to find the pathway for CLDN4 to prevention and treatment in laryngeal squamous cell carcinoma.

我们在前期工作中发现CLDN4基因在喉鳞状细胞癌组织及细胞中表达下调，并促进细胞恶性表型，且与DNA甲基化有关。本项目拟用ChIP、ChIP-reChIP、核酸酶可及性实验及RNAi等技术分析1)甲基化DNA与转录因子的结合2）MeCP2及组蛋白修饰在DNA甲基化下调CLDN4表达中的作用3）DNA甲基化对染色质构象的影响，揭示DNA甲基化下调喉癌细胞中CLDN4表达的机理；并拟通过细胞转染实验、跨上皮电阻的检测及Western blot等方法分析CLDN4过表达对1）紧密连接功能影响2）相关信号通路3）细胞迁移及侵袭相关蛋白表达的影响；最终揭示喉癌细胞中CLDN4表达下调及促进其恶性表型的机理,为CLDN4作为喉癌早期防治的靶标提供充分的理论基础。

课题组前期工作发现，DNA甲基化可以下调喉癌Hep-2细胞中CLDN4表达，并且可能与MeCP2蛋白有关。且CLDN4表达下调可以促进Hep-2细胞的恶性表型。.为进一步探讨DNA甲基化下调CLDN4表达的机制：采用亚硫酸氢盐测序（BSP）法检测Hep-2细胞中CLDN4启动子区中DNA甲基化位点，分析该区域的转录因子；采用免疫组织化学技术检测转录因子Sp1与CLDN4在喉癌组织中的表达；通过PCR技术及Western blot技术检测Sp1抑制剂MTM对CLDN4表达是否有调控作用；沉默MeCP2后检测喉癌Hep-2细胞中CLDN4的表达水平的变化；通过ChIP及Co-IP实验检测MeCP2与Sp1、HDAC1与基因组DNA的结合状态。结果显示：DNA甲基化可以抑制Sp1与CLDN4基因结合进而下调Hep-2细胞CLDN4表达；Hep-2细胞DNA甲基化下调CLDN4表达可能与MeCP2募集HDAC1有关。.为明确CLDN4表达改变影响喉癌Hep-2细胞恶性表型的机制：用免疫组织化学染色、Western-Blot及ELISA技术检测人喉癌组织以及Hep-2细胞中CLDN4、JNK-MMPs、JAK2/STAT3信号通路蛋白（P-JNK、MMP2、MMP9、JAK2、p-JAK2、STAT3和p-STAT3）的表达；细胞划痕、Transwell小室、CCK8、EdU和二维平板克隆形成实验检测各组细胞迁移、侵袭和增殖能力；采用透射电镜技术观察Hep-2细胞间紧密连接的变化。结果显示：CLDN4表达上调抑制Hep-2 细胞迁移和侵袭与JNK-MMPs通路有关；抑制细胞增殖与JAK2/STAT3信号通路有关；CLDN4表达上调可增加细胞间紧密连接强度。