【摘要】：Rap1是一種小分子GTP結(jié)合蛋白,它對(duì)于腫瘤細(xì)胞的極性、粘附和遷移能力都有重要調(diào)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(bào)道SIPA1在乳腺癌、結(jié)腸癌和前列腺等腫瘤中都異常高表達(dá),而且SIPA1的高表達(dá)會(huì)促進(jìn)腫瘤細(xì)胞的轉(zhuǎn)移。然而關(guān)于SIPA1為何在腫瘤中異常表達(dá),以及SIPA1是如何影響腫瘤轉(zhuǎn)移的機(jī)制并不清楚。DNA甲基化是一種調(diào)控基因轉(zhuǎn)錄的表觀修飾。DNA甲基化異常是腫瘤細(xì)胞區(qū)別與正常細(xì)胞的標(biāo)志之一。在腫瘤進(jìn)程中,抑癌基因的異常高甲基化會(huì)造成其表達(dá)沉默,而癌基因的異常低甲基化則激活其表達(dá)。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(zhuǎn)移惡化進(jìn)程中DNA甲基化也是動(dòng)態(tài)變化的。在腫瘤轉(zhuǎn)移進(jìn)程中CDH1基因的異常高甲基化,會(huì)導(dǎo)致E-cadherin的表達(dá)沉默,進(jìn)而通過(guò)下游信號(hào)通路的變化介導(dǎo)上皮間質(zhì)轉(zhuǎn)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(xì)胞中Sipa1失調(diào)的機(jī)理。通過(guò)檢測(cè)了不同類型腫瘤細(xì)胞(乳腺癌、結(jié)腸癌、前列腺癌和腦膠質(zhì)瘤)中的SIPA1的表達(dá),發(fā)現(xiàn)在轉(zhuǎn)移性強(qiáng)的腫瘤細(xì)胞中SIPA1表達(dá)普遍偏高。進(jìn)而通過(guò)甲基化特異性PCR,發(fā)現(xiàn)在細(xì)胞中Sipa1啟動(dòng)子區(qū)的CpG島的甲基化程度與SIPA1的蛋白水平呈負(fù)相關(guān)。對(duì)MDA-MB-361、Caco2細(xì)胞進(jìn)行藥物處理,發(fā)現(xiàn)甲基化抑制劑5-Aza-CdR和去乙酰化抑制劑TSA均能提升SIPA1的表達(dá),而且SIPA的表達(dá)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(bào)告基因?qū)嶒?yàn)證明:Sipa1啟動(dòng)子區(qū)的CpG島對(duì)于整個(gè)啟動(dòng)子的轉(zhuǎn)錄活性十分關(guān)鍵;當(dāng)CpG島被甲基化時(shí),Sipa1啟動(dòng)子的轉(zhuǎn)錄活性明顯下降。本研究還發(fā)現(xiàn)在乳腺癌細(xì)胞中SIPA1的高表達(dá)會(huì)抑制E-cadherin的表達(dá),進(jìn)而影響腫瘤細(xì)胞的上皮間質(zhì)轉(zhuǎn)換進(jìn)程。首先我們構(gòu)建了干擾SIPA1表達(dá)的231-sh-Sipa1細(xì)胞系,發(fā)現(xiàn)在該細(xì)胞系中E-cadherin表達(dá)被激活,vimentin表達(dá)則明顯下降;而在MCF7細(xì)胞過(guò)表達(dá)SIPA1,E-cadherin表達(dá)則下降。我們還研究了乳腺癌中SIPA1調(diào)控E-cadherin表達(dá)的機(jī)制。發(fā)現(xiàn)在MDA-MB-231細(xì)胞中,CDH1基因的呈中度甲基化;而在231-sh-Sipa1細(xì)胞系中,CDH1基因的甲基化水平則偏低。這說(shuō)明干擾SIPA1的表達(dá),會(huì)降低CDH1的甲基化水平。在MCF7細(xì)胞中的反向過(guò)表達(dá)SIPA1,CDH1基因的甲基化水平則升高。以上實(shí)驗(yàn)表明,在乳腺癌細(xì)胞中,SIPA1的異常高表達(dá)會(huì)提高CDH1基因的甲基化水平,進(jìn)而導(dǎo)致E-cadherin的表達(dá)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

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相關(guān)期刊論文 前1條

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