长链非编码RNA ANCR在乳腺癌侵袭和转移的作用和机制研究

发布时间：2018-04-01 03:11

本文选题：lncRNA　切入点：ANCR　出处：《东北师范大学》2017年博士论文

【摘要】：乳腺癌目前位列患者最多的几种癌症中,对人类的健康有很大影响。包括中国在内的许多国家和地区乳腺癌的死亡率逐年上升,其中由转移引发的乳腺癌患者死亡占所有死亡病例的90%以上,乳腺癌的恶性转移已经成为致死的重要原因。因此,深入研究乳腺癌转移的分子机制,对于发现乳腺癌治疗的新靶点,以及提高乳腺癌的治疗水平均具有十分重要的意义。现有的研究已经证实,癌症细胞经常会发生上皮-间质转化(EpithelialMesenchymal Transition,EMT),在包括乳腺癌的许多种恶性肿瘤患者的肿瘤细胞发生转移过程中发挥着重要作用。EMT的发生是多种信号通路、转录因子、表观修饰酶和非编码RNA等因素相互作用的结果。长链非编码RNA(long noncoding RNA,lncRNA)是指长度大于200 nt不具有蛋白编码功能的RNA分子。近年来的研究表明,lncRNA在表观遗传调控等多个层面上调控基因的表达,参与癌症细胞侵袭迁移等多种重要生命过程。lncRNA异常表达所引发的EMT变化与癌症的进程密切相关。近期的研究发现,lncRNA能够通过改变其结合蛋白的稳定性参与癌症进程。例如,在低氧微环境中,lncRNA-LET被组蛋白去乙酰化酶HDAC3下调后能增强NF-90蛋白泛素化降解,抑制癌细胞转移。EZH2(Enhancer of Zeste Homolog 2)是Polycomb抑制复合物2(PRC2)的一个亚基,在包括乳腺癌在内的多种癌症中高表达,并与癌症的进程和不良预后相关联。EZH2蛋白的稳定性也与其高水平存在有关。报道显示,EZH2的丝氨酸S75位发生糖基化修饰能够抑制EZH2泛素化降解,增强其蛋白稳定性;而CDK1介导的EZH2苏氨酸T345和T487位磷酸化修饰能促进其泛素化降解,降低EZH2蛋白的稳定性;EZH2赖氨酸K348位乙酰化修饰能够抑制EZH2泛素化降解,增强EZH2蛋白稳定性。在本文中研究中,我们首次发现在乳腺癌细胞中,lncRNA ANCR能够和EZH2结合,并且促进CDK1对EZH2磷酸化(T345和T487位),导致EZH2泛素化降解,最终降低其蛋白稳定性,即我们发现了一个调控EZH2蛋白稳定性的lncRNA ANCR。而且还进一步对lncRNA ANCR的功能进行了研究:首先发现ANCR在乳腺癌癌组织和乳腺癌细胞中均是低表达,敲低ANCR可以诱发MCF10A乳腺上皮细胞发生EMT,且能够促进MCF10A和MCF7乳腺癌细胞侵袭转移;在ANCR低表达的MDA-MB-231乳腺癌细胞中,我们发现过表达ANCR能够部分逆转EMT现象,且能够抑制其侵袭转移能力。其次,还发现过表达ANCR通过对EZH2稳定性的抑制还能够抑制EZH2的E-cadherin、HOXA10和DAB2IP等靶基因的表达;此外,还通过在过表达ANCR的细胞系中再过表达EZH2的回复实验,进一步确认ANCR在乳腺癌中的功能主要是通过抑制EZH2蛋白的稳定性来实现的。最后,通过裸鼠皮下注射和尾静脉注射检测ANCR对乳腺癌细胞体内的成瘤和肺转移能力的影响,结果证实ANCR在体内同样能够抑制乳腺癌细胞的成瘤能力和侵袭转移能力。因此,通过我们的体内体外细胞生物学和分子生物学实验,证实ANCR是一个能够抑制乳腺癌EMT和侵袭转移的lncRNA。我们的本次研究发现ANCR是一个能够抑制EZH2稳定性的lncRNA,还表明ANCR通过调控EZH2的蛋白稳定性能够抑制乳腺癌细胞EMT进程和乳腺癌侵袭转移的进程。这一新的发现预示着ANCR可能是一个乳腺癌潜在的诊断检测靶标,为乳腺癌的诊断和治疗提供新的思路。
[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.

【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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