本文選題：lncRNA　切入點：ANCR　出處：《東北師范大學(xué)》2017年博士論文

【摘要】：乳腺癌目前位列患者最多的幾種癌癥中,對人類的健康有很大影響。包括中國在內(nèi)的許多國家和地區(qū)乳腺癌的死亡率逐年上升,其中由轉(zhuǎn)移引發(fā)的乳腺癌患者死亡占所有死亡病例的90%以上,乳腺癌的惡性轉(zhuǎn)移已經(jīng)成為致死的重要原因。因此,深入研究乳腺癌轉(zhuǎn)移的分子機制,對于發(fā)現(xiàn)乳腺癌治療的新靶點,以及提高乳腺癌的治療水平均具有十分重要的意義�，F(xiàn)有的研究已經(jīng)證實,癌癥細(xì)胞經(jīng)常會發(fā)生上皮-間質(zhì)轉(zhuǎn)化(EpithelialMesenchymal Transition,EMT),在包括乳腺癌的許多種惡性腫瘤患者的腫瘤細(xì)胞發(fā)生轉(zhuǎn)移過程中發(fā)揮著重要作用。EMT的發(fā)生是多種信號通路、轉(zhuǎn)錄因子、表觀修飾酶和非編碼RNA等因素相互作用的結(jié)果。長鏈非編碼RNA(long noncoding RNA,lncRNA)是指長度大于200 nt不具有蛋白編碼功能的RNA分子。近年來的研究表明,lncRNA在表觀遺傳調(diào)控等多個層面上調(diào)控基因的表達,參與癌癥細(xì)胞侵襲遷移等多種重要生命過程。lncRNA異常表達所引發(fā)的EMT變化與癌癥的進程密切相關(guān)。近期的研究發(fā)現(xiàn),lncRNA能夠通過改變其結(jié)合蛋白的穩(wěn)定性參與癌癥進程。例如,在低氧微環(huán)境中,lncRNA-LET被組蛋白去乙�；窰DAC3下調(diào)后能增強NF-90蛋白泛素化降解,抑制癌細(xì)胞轉(zhuǎn)移。EZH2(Enhancer of Zeste Homolog 2)是Polycomb抑制復(fù)合物2(PRC2)的一個亞基,在包括乳腺癌在內(nèi)的多種癌癥中高表達,并與癌癥的進程和不良預(yù)后相關(guān)聯(lián)。EZH2蛋白的穩(wěn)定性也與其高水平存在有關(guān)。報道顯示,EZH2的絲氨酸S75位發(fā)生糖基化修飾能夠抑制EZH2泛素化降解,增強其蛋白穩(wěn)定性;而CDK1介導(dǎo)的EZH2蘇氨酸T345和T487位磷酸化修飾能促進其泛素化降解,降低EZH2蛋白的穩(wěn)定性;EZH2賴氨酸K348位乙�；揎椖軌蛞种艵ZH2泛素化降解,增強EZH2蛋白穩(wěn)定性。在本文中研究中,我們首次發(fā)現(xiàn)在乳腺癌細(xì)胞中,lncRNA ANCR能夠和EZH2結(jié)合,并且促進CDK1對EZH2磷酸化(T345和T487位),導(dǎo)致EZH2泛素化降解,最終降低其蛋白穩(wěn)定性,即我們發(fā)現(xiàn)了一個調(diào)控EZH2蛋白穩(wěn)定性的lncRNA ANCR。而且還進一步對lncRNA ANCR的功能進行了研究:首先發(fā)現(xiàn)ANCR在乳腺癌癌組織和乳腺癌細(xì)胞中均是低表達,敲低ANCR可以誘發(fā)MCF10A乳腺上皮細(xì)胞發(fā)生EMT,且能夠促進MCF10A和MCF7乳腺癌細(xì)胞侵襲轉(zhuǎn)移;在ANCR低表達的MDA-MB-231乳腺癌細(xì)胞中,我們發(fā)現(xiàn)過表達ANCR能夠部分逆轉(zhuǎn)EMT現(xiàn)象,且能夠抑制其侵襲轉(zhuǎn)移能力。其次,還發(fā)現(xiàn)過表達ANCR通過對EZH2穩(wěn)定性的抑制還能夠抑制EZH2的E-cadherin、HOXA10和DAB2IP等靶基因的表達;此外,還通過在過表達ANCR的細(xì)胞系中再過表達EZH2的回復(fù)實驗,進一步確認(rèn)ANCR在乳腺癌中的功能主要是通過抑制EZH2蛋白的穩(wěn)定性來實現(xiàn)的。最后,通過裸鼠皮下注射和尾靜脈注射檢測ANCR對乳腺癌細(xì)胞體內(nèi)的成瘤和肺轉(zhuǎn)移能力的影響,結(jié)果證實ANCR在體內(nèi)同樣能夠抑制乳腺癌細(xì)胞的成瘤能力和侵襲轉(zhuǎn)移能力。因此,通過我們的體內(nèi)體外細(xì)胞生物學(xué)和分子生物學(xué)實驗,證實ANCR是一個能夠抑制乳腺癌EMT和侵襲轉(zhuǎn)移的lncRNA。我們的本次研究發(fā)現(xiàn)ANCR是一個能夠抑制EZH2穩(wěn)定性的lncRNA,還表明ANCR通過調(diào)控EZH2的蛋白穩(wěn)定性能夠抑制乳腺癌細(xì)胞EMT進程和乳腺癌侵襲轉(zhuǎn)移的進程。這一新的發(fā)現(xiàn)預(yù)示著ANCR可能是一個乳腺癌潛在的診斷檢測靶標(biāo),為乳腺癌的診斷和治療提供新的思路。
[Abstract]:At present most of the breast cancer in several cancer patients, has great influence on human health. Chinese, including many countries and regions, the mortality rate of breast cancer increased year by year, which caused by metastasis in patients with breast cancer deaths accounted for more than 90% of all deaths, malignant metastatic breast cancer has become an important cause of death. So and the further study of molecular mechanism of metastasis of breast cancer, to find new targets for the treatment of breast cancer, and improve the level of treatment of breast cancer is very important. The existing research has confirmed that cancer cells often occurs in epithelial mesenchymal transition (EpithelialMesenchymal Transition, EMT), occurs in many kinds of malignant tumors in patients with breast cancer including tumor cells play an important role in the occurrence of.EMT is multiple signaling pathways, transcription factor in the transfer process, the apparent modification of enzyme and non encoding The interaction of RNA and other factors. The results of long chain non encoding RNA (long noncoding RNA, lncRNA RNA) is a molecular length greater than 200 NT has no protein encoding function. Recent studies show that lncRNA in epigenetic regulation, gene expression and regulation on a lot of aspects involved in cancer cell invasion and migration, etc. many important life processes of the abnormal expression of.LncRNA and changes of EMT caused by cancer is closely related to the process. A recent study showed that lncRNA can change its stability by binding proteins involved in cancer process. For example, in the hypoxic microenvironment, lncRNA-LET histone deacetylase HDAC3 downregulation can enhance NF-90 protein ubiquitination. Inhibition of cancer cell metastasis of.EZH2 (Enhancer of Zeste Homolog 2) is the inhibition of Polycomb complex 2 (PRC2) is a subunit, highly expressed in many cancers including breast cancer, and cancer progression Stability and poor prognosis of.EZH2 associated protein with high levels of existence. Reports indicate that EZH2 S75 a serine glycosylation could inhibit EZH2 ubiquitination, increase its protein stability; CDK1 mediated EZH2 T345 and T487 threonine phosphorylation can promote its ubiquitination, reducing stability EZH2 protein; EZH2 K348 lysine acetylation modification can inhibit EZH2 ubiquitination, enhanced EZH2 protein stability. In this paper, we first found in breast cancer cells, lncRNA ANCR can be combined with EZH2, CDK1 and promote the phosphorylation of EZH2 (T345 and T487), resulting in EZH2 ubiquitin degradation, and ultimately reduce the protein stability, we found a regulating EZH2 protein stability of lncRNA ANCR. on lncRNA ANCR and further studied the function of ANCR in breast cancer: first discovered Cancer and breast cancer cells were low expression, knockdown of ANCR can induce MCF10A mammary epithelial cells EMT, and MCF10A and MCF7 can promote the invasion and metastasis of breast cancer cells; in the low expression of ANCR MDA-MB-231 in breast cancer cells, we found that overexpression of ANCR could partially reverse the EMT phenomenon, and can inhibit the invasion transfer ability. Secondly, also found that overexpression of ANCR can inhibit EZH2 through the inhibition on the stability of EZH2 E-cadherin, the expression of HOXA10 and DAB2IP target genes; in addition, the expression of ANCR cell lines and expression recovery experiment EZH2, further confirmed the ANCR function in breast cancer is mainly to achieve stability through inhibition of EZH2 protein. Finally, the effects of subcutaneous injection and intravenous injection of ANCR in vivo detection of breast cancer cells into tumor and lung metastasis, the results demonstrated that ANCR in vivo Can also inhibit breast cancer cell tumorigenicity and metastasis. Therefore, through our in vitro and in vivo cell biology and molecular biology experiments demonstrate that ANCR is a can inhibit the invasion and metastasis of breast cancer EMT and lncRNA. in this study we found that ANCR is a can inhibit the stability of EZH2 lncRNA, also showed that ANCR through the regulation of protein stability of EZH2 can inhibit the breast cancer cell EMT and the process of the invasion and metastasis of breast cancer progression. This new discovery indicates that ANCR may be a potential target for breast cancer diagnosis, and provide a new way for the diagnosis and treatment of breast cancer.

【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.9

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本文編號：1693689