【作者】 刘亚芳；

【导师】 全成实；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 目的:探讨乳腺癌细胞MCF-7中雌激素受体α(estrogen receptor alpha, ERα)和DNA甲基化介导CLDN6表达的作用机制及其对MCF-7细胞转移表型的影响。方法:采用RT-PCR、Western blot及细胞免疫荧光法检测ERα激动剂(propyl pyrazole triol,PPT)和雌激素受体(estrogen receptor, ER)阻断剂ICI 182,780(ICI)对CLDN6表达的影响;采用Western blot和甲基化特异性PCR(methylation specific PCR,MSP)法检测乳腺癌组织中CLDN6表达和DNA甲基化状态并分析其相互关系;利用5-aza-dC和TSA作用MCF-7细胞,采用RT-PCR、Western blot、细胞免疫荧光法及MSP法检测乳腺癌细胞中CLDN6表达和DNA甲基化状态,用染色质免疫沉淀技术(Chromatin immunoprecitation,ChIP)检测CLDN6基因与甲基化CpG位点结合蛋白2(5 methyl-CpG binding protein 2,MeCP2)、乙酰化的组蛋白(acetylated H3,H3Ac;acetylated H4,H4Ac)结合;用核酸酶可及性实验分析CLDN6基因所在区域染色质构象;用RNAi技术分析沉默MeCP2对CLDN6表达的影响;划痕法和Transwell小室分析DNA甲基化下调CLDN6表达对MCF-7迁移和侵袭表型的影响。结果:PPT能够上调MCF-7细胞CLDN6的表达,ICI能够阻断PPT对CLDN6表达的诱导作用;乳腺癌组织和细胞中CLDN6低表达,并与DNA甲基化有关;5-aza-dC和TSA单独及联合作用MCF-7细胞能够上调CLDN6表达;5-aza-dC作用MCF-7细胞可以抑制MeCP2与CLDN6基因的结合,促进H3Ac和H4Ac与CLDN6基因的结合,TSA可以促进H4Ac与CLDN6基因的结合;5-aza-dC可以使MCF-7细胞中CLDN6基因CpG岛附近染色质结构变疏松;RNAi技术沉默MeCP2对CLDN6表达无明显影响;5-aza-dC可抑制MCF-7细胞的侵袭和迁移能力,沉默5-aza-dC作用组细胞中的CLDN6基因能够增强细胞的迁移和侵袭能力。结论:ERα可以上调MCF-7细胞中CLDN6表达;乳腺癌组织和细胞中CLDN6表达下调与DNA甲基化有关;DNA甲基化下调CLDN6表达与MeCP2的结合、组蛋白H3和H4脱乙酰化及染色质构象改变有关;DNA甲基化下调CLDN6表达可以促进MCF-7细胞的转移表型。更多还原

【Abstract】 Tight junctions (TJ) are located at the extreme apical region of junction-associated complexes in epithelial and endothelial cells, where they play important roles in maintaining cell polarity, cell adherence and regulating cell proliferation and differentiation. Claudins (CLDNs) are the main components of tight junctions, forming the backbone of tight junction strands and transmitting cellular signals. Their expression has been reported altered in several cancers. For example, CLDN1 was downregulated in breast cancer and colon cancer, CLDN7 was reduced in invasive breast cancer , CLDN3 and CLDN4 have been reported to be upregulated in ovarian cancer. Whether upregulated or downregulated, the functions and structure of tight junctions are often abnormal in a number of cancers .CLDN6, one of 24 members of the CLDN family, it is located on chromosome 16 p13.3,encodes a 23 kDa membrane protein of 219 amino acids. Recent reports show that CLDN6 can regulate adipogenesis and fat deposition , it is required for normal blastocyst formation, it is important for maintaining permeability barriers and transepithelial resistance of epidermal cells. In previous work, we found that the expression of CLDN6 was down-regulated in human and rat mammary cancer cell lines. MCF-7 cells transfected with CLDN6 grew slowly, anchorage-independent growth, invasive and migratory traits were also substantially decreased in cells with CLDN6 expression. These results suggest that CLDN6 may act as a cancer suppressor.According to our previous work, CLDN6 expression was related to ERαin breast cancer tissues and 17β-estradiol(17β-E2)could up-regulate the expression of CLDN6 in MCF-7 cells. According to current reports, ERαmight regulate the expression of CLDN1, 3, 4 in breast cancer tissues. But, the roles of ERαin the regulation of expression of CLDN6 in MCF-7 cells is unclear.According to current reports, DNA methylation could down-regulate CLDNs expression. DNA methylation leads to transcriptional repression either by directly interfering with the binding of transcription factors or by the involvement of proteins that specifically recognize methylated CpGs, the prototype of which is the methyl-CpG-binding protein 2 (MeCP2); MeCP2 specifically targets mCpGs and determines repressive heterochromatin by interacting with histone deacetylases and histone methyltransferase; DNA methylation could also repress gene transcription by mechanisms of chromatin condensation. There are some reports that DNA methylation could down-regulate CLDN6 expression in breast cancer cells and esophageal carcinoma tissues. However, the mechanisms of 17-β-estradiol and DNA methylation regulating the expression of CLDN6 are still unclear.This study sought to determine the role of ERs in the regulation of expression of CLDN6 and the mechanism of DNA methylation regulating the expression of CLDN6.Methods:1. The effects of ERαon CLDN6 expression: PPT and ICI treatment were performed. The expression of CLDN6 was measured by reverse transcriptase-PCR (RT-PCR), western blotting and immunofluorescent staining.2. Expression of CLDN6 and DNA methylation : RT-PCR, western blotting, immunofluorescent staining were used to determine the expression of CLDN6 in breast cancer tissues and control tissues and breast cancer cells without or with 5-Aza-2’-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) treated; Methylation-Specific PCR (MSP) was used to determine methylation status at the CLDN6 gene.3. The machanism DNA methylation regulating the expression of CLDN6: ChIP was used to determine the combination of CLDN6 with MeCP2, H3Ac and H4Ac protein; Nuclease accessibility assays was performed to obtain information about the nucleosomal organization of the CLDN6 gene. To confirm the role of MeCP2 in the regulation of CLDN6 expression in MCF-7 cells, we used RNAi to knockdown MeCP2 in MCF-7 cells.4. The effects DNA methylation of CLDN6 gene on metastasis phaenotype of MCF-7: Wound healing assay and transwell assay were used to determine the metastasis phaenotype of MCF-7.Results:1. The effects of ERαon CLDN6 expression: PPT could upregulate the expression of CLDN6, and the effect could be blocked by ICI.2. Expression of CLDN6 and DNA methylation:The expression of CLDN6 in breast cancer was lower than control group, lymphode metastasis group was lower than control group. DNA methylation of CLDN6 in breast cancer was higher than control group. MCF-7 cells did not express CLDN6 in accord with hypermethylation at the CLDN6 CpG sites, 5-aza-dC and TSA could induce the expression of CLDN6 separately or simultaneously. And 5-aza-dC could increase CLDN6 demethylation status. 3. The machanism DNA methylation regulating the expression of CLDN6: 5-aza-dC can inhibit the combination of CLDN6 with MeCP2, H3Ac and H4Ac; After treating MCF-7 with 5-aza–dC, the chromatin structure nearby CLDN6 gene became more loose than control group; MeCP2 silence have no effection on the expression of CLDN6.4. The effects of CLDN6 gene DNA methylation on metastasis phaenotype of MCF-7: 5-aza-dC could suppress migration and metastatic ability of MCF-7; Slience expression of CLDN6 in the 5-aza-dC treated group would enhance the migration and metastatic ability.Conclusions:1. ERαcould upregulate the expression of CLDN6.2. CLDN6 expression was related to DNA methylation in breast cancer tissues and cells.3. DNA methylation down-regulated CLDN6 expression releated to the combination of CLDN6 with MeCP2, H3, H4 deacetylation and the chromatin structure alteration.4. DNA methylation down-regulated CLDN6 expression would enhance migration phenotype of MCF-7 cells.更多还原