乳腺癌细胞中SIPA-1失调的表观遗传学机制及其对E-cadherin表达调控的初步研究
发布时间：2018-10-09 15:08
【摘要】：Rap1是一种小分子GTP结合蛋白,它对于肿瘤细胞的极性、粘附和迁移能力都有重要调节作用。SIPA1是Rap1的GTP水解蛋白,它催化活性形式Rap1GTP水解变为失活形式的Rap1GDP。已报道SIPA1在乳腺癌、结肠癌和前列腺等肿瘤中都异常高表达,而且SIPA1的高表达会促进肿瘤细胞的转移。然而关于SIPA1为何在肿瘤中异常表达,以及SIPA1是如何影响肿瘤转移的机制并不清楚。DNA甲基化是一种调控基因转录的表观修饰。DNA甲基化异常是肿瘤细胞区别与正常细胞的标志之一。在肿瘤进程中,抑癌基因的异常高甲基化会造成其表达沉默,而癌基因的异常低甲基化则激活其表达。DNA甲基化异常的发生要早于肿瘤的初始形成,而且在肿瘤的转移恶化进程中DNA甲基化也是动态变化的。在肿瘤转移进程中CDH1基因的异常高甲基化,会导致E-cadherin的表达沉默,进而通过下游信号通路的变化介导上皮间质转换的发生。在本研究中,我们首先从DNA甲基化的角度,分析了在肿瘤细胞中Sipa1失调的机理。通过检测了不同类型肿瘤细胞(乳腺癌、结肠癌、前列腺癌和脑胶质瘤)中的SIPA1的表达,发现在转移性强的肿瘤细胞中SIPA1表达普遍偏高。进而通过甲基化特异性PCR,发现在细胞中Sipa1启动子区的CpG岛的甲基化程度与SIPA1的蛋白水平呈负相关。对MDA-MB-361、Caco2细胞进行药物处理,发现甲基化抑制剂5-Aza-CdR和去乙酰化抑制剂TSA均能提升SIPA1的表达,而且SIPA的表达水平与5-Aza-dC的浓度呈依赖性。更为重要的是,体外甲基化荧光素酶报告基因实验证明:Sipa1启动子区的CpG岛对于整个启动子的转录活性十分关键;当CpG岛被甲基化时,Sipa1启动子的转录活性明显下降。本研究还发现在乳腺癌细胞中SIPA1的高表达会抑制E-cadherin的表达,进而影响肿瘤细胞的上皮间质转换进程。首先我们构建了干扰SIPA1表达的231-sh-Sipa1细胞系,发现在该细胞系中E-cadherin表达被激活,vimentin表达则明显下降;而在MCF7细胞过表达SIPA1,E-cadherin表达则下降。我们还研究了乳腺癌中SIPA1调控E-cadherin表达的机制。发现在MDA-MB-231细胞中,CDH1基因的呈中度甲基化;而在231-sh-Sipa1细胞系中,CDH1基因的甲基化水平则偏低。这说明干扰SIPA1的表达,会降低CDH1的甲基化水平。在MCF7细胞中的反向过表达SIPA1,CDH1基因的甲基化水平则升高。以上实验表明,在乳腺癌细胞中,SIPA1的异常高表达会提高CDH1基因的甲基化水平,进而导致E-cadherin的表达沉默。
[Abstract]:Rap1 is a small molecule GTP binding protein, which plays an important role in regulating the polarity, adhesion and migration of tumor cells. SIPA1 is the GTP hydrolysate of Rap1. It catalyzes the hydrolysis of Rap1GTP into inactivated Rap1GDP.. It has been reported that SIPA1 is highly expressed in breast cancer, colon cancer and prostate cancer, and the high expression of SIPA1 can promote the metastasis of tumor cells. However, it is not clear that the abnormal expression of SIPA1 in tumor and the mechanism of SIPA1 affecting tumor metastasis. DNA methylation is an epigenetic modification of gene transcription.DNA methylation abnormality is one of the markers of differentiating tumor cells from normal cells. The abnormal hypermethylation of the tumor suppressor gene causes its expression silencing, while the abnormal hypomethylation of the oncogene activates the abnormal expression of .DNA methylation earlier than the initial formation of the tumor. Moreover, DNA methylation is also dynamic in the process of metastasis and deterioration of tumor. The abnormal hypermethylation of CDH1 gene in the process of tumor metastasis leads to the silencing of E-cadherin expression, which in turn mediates epithelial mesenchymal transition through the change of downstream signaling pathway. In this study, we first analyzed the mechanism of Sipa1 imbalance in tumor cells from the point of view of DNA methylation. By detecting the expression of SIPA1 in different types of tumor cells (breast, colon, prostate and glioma), we found that the expression of SIPA1 was generally higher in metastatic tumor cells. The methylation degree of CpG island in the Sipa1 promoter was negatively correlated with the protein level of SIPA1 by methylation-specific PCR,. It was found that both methylation inhibitor 5-Aza-CdR and deacetylation inhibitor TSA could enhance the expression of SIPA1 in MDA-MB-361,Caco2 cells, and the expression level of SIPA was dependent on the concentration of 5-Aza-dC. More importantly, the in vitro methylation luciferase reporter gene experiment showed that the CpG island of the 1: Sipa1 promoter was critical to the transcriptional activity of the whole promoter, and that the transcriptional activity of the Sipa1 promoter decreased significantly when the CpG island was methylated. We also found that the high expression of SIPA1 in breast cancer cells inhibited the expression of E-cadherin and affected the process of epithelial interstitial transition. Firstly, we constructed a 231-sh-Sipa1 cell line that interfered with the expression of SIPA1. It was found that the expression of E-cadherin was significantly decreased in this cell line, but the expression of SIPA1,E-cadherin was decreased in MCF7 cells. We also studied the mechanism of E-cadherin expression regulated by SIPA1 in breast cancer. It was found that the methylation of CDH1 gene in MDA-MB-231 cells was moderate, but the methylation level of CDH1 gene in 231-sh-Sipa1 cell line was lower than that in 231-sh-Sipa1 cell line. This suggests that interfering with the expression of SIPA1 reduces the methylation level of CDH1. The methylation level of overexpression of SIPA1,CDH1 gene in MCF7 cells was increased. These results suggest that the abnormal high expression of siPA1 in breast cancer cells can increase the methylation level of CDH1 gene and lead to the silencing of E-cadherin expression.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
本文编号：2259792
[Abstract]:Rap1 is a small molecule GTP binding protein, which plays an important role in regulating the polarity, adhesion and migration of tumor cells. SIPA1 is the GTP hydrolysate of Rap1. It catalyzes the hydrolysis of Rap1GTP into inactivated Rap1GDP.. It has been reported that SIPA1 is highly expressed in breast cancer, colon cancer and prostate cancer, and the high expression of SIPA1 can promote the metastasis of tumor cells. However, it is not clear that the abnormal expression of SIPA1 in tumor and the mechanism of SIPA1 affecting tumor metastasis. DNA methylation is an epigenetic modification of gene transcription.DNA methylation abnormality is one of the markers of differentiating tumor cells from normal cells. The abnormal hypermethylation of the tumor suppressor gene causes its expression silencing, while the abnormal hypomethylation of the oncogene activates the abnormal expression of .DNA methylation earlier than the initial formation of the tumor. Moreover, DNA methylation is also dynamic in the process of metastasis and deterioration of tumor. The abnormal hypermethylation of CDH1 gene in the process of tumor metastasis leads to the silencing of E-cadherin expression, which in turn mediates epithelial mesenchymal transition through the change of downstream signaling pathway. In this study, we first analyzed the mechanism of Sipa1 imbalance in tumor cells from the point of view of DNA methylation. By detecting the expression of SIPA1 in different types of tumor cells (breast, colon, prostate and glioma), we found that the expression of SIPA1 was generally higher in metastatic tumor cells. The methylation degree of CpG island in the Sipa1 promoter was negatively correlated with the protein level of SIPA1 by methylation-specific PCR,. It was found that both methylation inhibitor 5-Aza-CdR and deacetylation inhibitor TSA could enhance the expression of SIPA1 in MDA-MB-361,Caco2 cells, and the expression level of SIPA was dependent on the concentration of 5-Aza-dC. More importantly, the in vitro methylation luciferase reporter gene experiment showed that the CpG island of the 1: Sipa1 promoter was critical to the transcriptional activity of the whole promoter, and that the transcriptional activity of the Sipa1 promoter decreased significantly when the CpG island was methylated. We also found that the high expression of SIPA1 in breast cancer cells inhibited the expression of E-cadherin and affected the process of epithelial interstitial transition. Firstly, we constructed a 231-sh-Sipa1 cell line that interfered with the expression of SIPA1. It was found that the expression of E-cadherin was significantly decreased in this cell line, but the expression of SIPA1,E-cadherin was decreased in MCF7 cells. We also studied the mechanism of E-cadherin expression regulated by SIPA1 in breast cancer. It was found that the methylation of CDH1 gene in MDA-MB-231 cells was moderate, but the methylation level of CDH1 gene in 231-sh-Sipa1 cell line was lower than that in 231-sh-Sipa1 cell line. This suggests that interfering with the expression of SIPA1 reduces the methylation level of CDH1. The methylation level of overexpression of SIPA1,CDH1 gene in MCF7 cells was increased. These results suggest that the abnormal high expression of siPA1 in breast cancer cells can increase the methylation level of CDH1 gene and lead to the silencing of E-cadherin expression.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9
【参考文献】
相关期刊论文 前1条
1 李晓军;赵阳;任宏;;遗传性和散发性胃癌上皮型钙黏附素的表达和启动子甲基化状态的关系[J];南方医科大学学报;2013年01期
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