乳腺癌細胞中SIPA-1失調的表觀遺傳學機制及其對E-cadherin表達調控的初步研究
發(fā)布時間：2018-10-09 15:08
【摘要】：Rap1是一種小分子GTP結合蛋白,它對于腫瘤細胞的極性、粘附和遷移能力都有重要調節(jié)作用。SIPA1是Rap1的GTP水解蛋白,它催化活性形式Rap1GTP水解變?yōu)槭Щ钚问降腞ap1GDP。已報道SIPA1在乳腺癌、結腸癌和前列腺等腫瘤中都異常高表達,而且SIPA1的高表達會促進腫瘤細胞的轉移。然而關于SIPA1為何在腫瘤中異常表達,以及SIPA1是如何影響腫瘤轉移的機制并不清楚。DNA甲基化是一種調控基因轉錄的表觀修飾。DNA甲基化異常是腫瘤細胞區(qū)別與正常細胞的標志之一。在腫瘤進程中,抑癌基因的異常高甲基化會造成其表達沉默,而癌基因的異常低甲基化則激活其表達。DNA甲基化異常的發(fā)生要早于腫瘤的初始形成,而且在腫瘤的轉移惡化進程中DNA甲基化也是動態(tài)變化的。在腫瘤轉移進程中CDH1基因的異常高甲基化,會導致E-cadherin的表達沉默,進而通過下游信號通路的變化介導上皮間質轉換的發(fā)生。在本研究中,我們首先從DNA甲基化的角度,分析了在腫瘤細胞中Sipa1失調的機理。通過檢測了不同類型腫瘤細胞(乳腺癌、結腸癌、前列腺癌和腦膠質瘤)中的SIPA1的表達,發(fā)現(xiàn)在轉移性強的腫瘤細胞中SIPA1表達普遍偏高。進而通過甲基化特異性PCR,發(fā)現(xiàn)在細胞中Sipa1啟動子區(qū)的CpG島的甲基化程度與SIPA1的蛋白水平呈負相關。對MDA-MB-361、Caco2細胞進行藥物處理,發(fā)現(xiàn)甲基化抑制劑5-Aza-CdR和去乙�；种苿㏕SA均能提升SIPA1的表達,而且SIPA的表達水平與5-Aza-dC的濃度呈依賴性。更為重要的是,體外甲基化熒光素酶報告基因實驗證明:Sipa1啟動子區(qū)的CpG島對于整個啟動子的轉錄活性十分關鍵;當CpG島被甲基化時,Sipa1啟動子的轉錄活性明顯下降。本研究還發(fā)現(xiàn)在乳腺癌細胞中SIPA1的高表達會抑制E-cadherin的表達,進而影響腫瘤細胞的上皮間質轉換進程。首先我們構建了干擾SIPA1表達的231-sh-Sipa1細胞系,發(fā)現(xiàn)在該細胞系中E-cadherin表達被激活,vimentin表達則明顯下降;而在MCF7細胞過表達SIPA1,E-cadherin表達則下降。我們還研究了乳腺癌中SIPA1調控E-cadherin表達的機制。發(fā)現(xiàn)在MDA-MB-231細胞中,CDH1基因的呈中度甲基化;而在231-sh-Sipa1細胞系中,CDH1基因的甲基化水平則偏低。這說明干擾SIPA1的表達,會降低CDH1的甲基化水平。在MCF7細胞中的反向過表達SIPA1,CDH1基因的甲基化水平則升高。以上實驗表明,在乳腺癌細胞中,SIPA1的異常高表達會提高CDH1基因的甲基化水平,進而導致E-cadherin的表達沉默。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R737.9
本文編號：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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R737.9
【參考文獻】
相關期刊論文 前1條
1 李曉軍;趙陽;任宏;;遺傳性和散發(fā)性胃癌上皮型鈣黏附素的表達和啟動子甲基化狀態(tài)的關系[J];南方醫(yī)科大學學報;2013年01期
，本文編號：2259792
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