肝癌干细胞相关基因和长链非编码RNA调控自我更新的分子机制研究

发布时间：2018-06-04 12:15

本文选题：肝癌 + 肿瘤干细胞　；参考：《中国科学技术大学》2016年博士论文

【摘要】：原发性肝癌是全球五大常见癌症之一,中国每年有超过46万新发病例和42万死亡患者,肝癌被称为我国的“国病”,严重威胁着人们的健康水平。肿瘤干细胞学说认为在肿瘤组织中,只有极少量的致瘤性细胞亚群具有无限增生的潜能,具有自我更新和多向分化能力,产生异质性肿瘤细胞,且对化疗药物有耐药性。肿瘤干细胞在启始肿瘤形成和生长中起着关键作用,目前的治疗措施还无法针对肿瘤干细胞发挥作用,这可能是导致多种肿瘤复发、转移和耐药的主要原因。因此,若要治愈肿瘤,必须清除肿瘤中的干细胞。但是肝癌干细胞的自我更新机制仍需进一步阐明。我们前期研究发现,CD133和CD13两个分子的联合应用能够分选出干性能力更强的肝癌干细胞。我们分选出肝癌干细胞(CD133+CD13+)和非干细胞(CD133-CD13-),做转录组芯片分析,鉴定了在肿瘤干细胞中起重要调节作用的基因(C8orf4和Zic2)与长链非编码RNA (Lnc-β-Catm和LncBrm),并系统研究了它们对肝癌干细胞自我更新的调控作用及其分子机制。转录组分析和临床样本验证均显示C8orf4在肝癌和肝癌干细胞中低表达。C8orf4敲低之后,肝癌干细胞的小球形成能力和肿瘤起始能力削弱。进一步研究发现,C8o1f4负调控Notch信号,并得到了临床数据的支持。通过酵母双杂交筛选,我们发现C8orf4与N2ICD (Notch2胞内段)有相互作用,实验证实C8orf4影响N2ICD的入核而N2ICD的入核对Notch2信号的活化至关重要。进一步研究表明,Notch2本身及其下游基因Nrarp、Hey1等在肝癌干细胞中也起着非常重要的作用,Notch2、Nrarp和Heyl敲低的细胞有较低的小球形成能力和肿瘤起始能力。还发现Notch靶基因的表达量和肝癌患者的临床分期分级、临床严重性和预后有关。Zic2在肝癌中高表达,在肝癌干细胞中表达量更高。研究表明Zic2敲除的细胞形成小球能力和肿瘤起始能力变弱,Zic2高表达的细胞干性增强。检测发现,Zic2敲除的细胞中OCT4及其下游基因表达量变低,而Zic2高表达的细胞中OCT4表达量升高,证实Zic2能够影响OCT4的表达。酵母双杂交的结果显示,Zic2能够结合NURF复合物,而N JRF复合物对于OCT4启动子区的染色体开放和启动转录至关重要。总之,Zic2通过招募NURF复合物,启动OCT4的表达,继而参与肝癌干细胞自我更新的维持。长链非编码RNA (lncRNA)是一类重要的调节分子,在基因表达和其它多种生物学过程中发挥重要作用。我们发现,Lnc-β-Catm分布于细胞核中,调节肝癌干细胞的小球形成能力和肿瘤起始能力。Lnc-β-Catm敲低的细胞干性削弱,而高表达Lnc-β-Catm的细胞干性增强。进一步研究发现,Lnc-β-Catm影响Wnt/β-cateni n信号通路。Lnc-β-Catm能够结合β-catenin和EZH2, EZH2能够甲基化β-catenin,甲基化的P-catenin稳定性增加。通过临床样本的检测,发现Lnc-β-Catm的表达量和Wnt/β-catenin的活性与肝癌的严重程度和预后相关。综上所述,Lnc-β-Catm能够促进β-catenin和EZH2的相互作用,进而促进β-catenin的甲基化,增强了β-catenin的稳定性,从而活化了Wnt/β-catenin信号通路,在肝癌干细胞自我更新维持中发挥重要作用。我们还发现,LncBrm在肝癌干细胞中高表达并促进其自我更新。LncBrm敲低的细胞中,Yap1信号通路受到抑制,而LncBrm高表达则促进Yap1的活化。进一步研究发现,LncBrm与Brm相互作用,并抑制Brm类型的SWI/SNF复合物的组装,促进Brg1类型的SWI/SNF复合物的组装。Brg1类型的复合物能够通过招募K1f4结合到Yap1的启动子区域,启动Yap1的表达,Yap1信号在促进了肝癌干细胞自我更新的维持。总之,我们鉴定了在肿瘤干细胞中起重要调节作用的基因(C8orf4 和 Zic2)与lncRNA(Lnc-β-Catm和LncBrm),确定了它们对肝癌干细胞自我更新的影响及其揭示了其调控自我更新的分子机制。
[Abstract]:Primary liver cancer is one of the five most common cancers in the world. There are more than 46 million new cases and 420 thousand deaths in China each year. Liver cancer is known as "national disease" in China. It is a serious threat to people's health. The tumor stem cell theory holds that only a few tumor cells in tumor stem cells have the potential of unlimited proliferation. The cancer stem cells play a key role in the initiation and growth of tumor, and the current treatment measures can not play a role in the cancer stem cells. This may be the main cause of the recurrence, metastasis and resistance of various tumors. In order to cure the tumor, the stem cells in the tumor must be removed. But the self-renewal mechanism of the liver cancer stem cells still needs to be further clarified. Our previous study found that the combination of the two molecules of CD133 and CD13 could be used to separate the stronger stem cells of liver cancer. We were able to separate the liver cancer stem cells (CD133+CD13+) and non stem cells (CD1 33-CD13-), the genes (C8orf4 and Zic2) and long chain non coded RNA (Lnc- beta -Catm and LncBrm) which play important roles in tumor stem cells were identified by transcriptional chip analysis, and their regulation and molecular mechanism on the self-renewal of liver cancer stem cells were systematically studied. Both transcriptional and clinical samples showed that C8orf4 was in the system. After the low expression of.C8orf4 in the liver and liver cancer stem cells, the formation ability of the liver cancer stem cells and the tumor initiation ability were weakened. Further studies found that C8o1f4 negatively regulated the Notch signal and was supported by clinical data. We found that the interaction between C8orf4 and N2ICD (Notch2 intracellular segment) was tested by yeast two hybrid screening. It is confirmed that C8orf4 affects the nucleation of N2ICD and the activation of N2ICD's Notch2 signal is crucial. Further studies have shown that Notch2 itself and its downstream gene Nrarp, Hey1, etc. play a very important role in the stem cells of liver cancer. Notch2, Nrarp and Heyl knockout cells have lower pellet formation and tumor initiation. The expression of otch target gene and the clinical staging of liver cancer patients, the clinical severity and prognosis related to the high expression of.Zic2 in the liver cancer and higher expression in the liver cancer stem cells. The study shows that the ability of Zic2 knockout cells to form the small ball and the tumor initiation ability and the enhancement of the Zic2 high expression of the cells. Detection and detection of Zic2 knockout The expression of OCT4 and its downstream genes in the cell was low, and the expression of OCT4 in Zic2 high expressed cells increased, which confirmed that Zic2 could affect the expression of OCT4. The results of yeast two hybrid showed that Zic2 could combine NURF complexes, and N JRF complex was essential for the opening and transcription of chromosomes in the OCT4 promoter region. RF complex, which starts the expression of OCT4, and then participates in the maintenance of self renewal of liver cancer stem cells. Long chain non coded RNA (lncRNA) is an important class of regulators and plays an important role in gene expression and other biological processes. We found that Lnc- beta -Catm is distributed in the nucleus of the cell to regulate the pellet formation of liver cancer stem cells. The tumor initiation ability of.Lnc- beta -Catm knockdown cells is weakened, and the cells with high expression of Lnc- beta -Catm are enhanced. Further studies have found that Lnc- beta -Catm affects the Wnt/ beta -cateni n signaling pathway.Lnc- beta -Catm can be combined with beta -catenin and -Catm, and the stability of methylation is increased. It is found that the expression of Lnc- beta -Catm and the activity of Wnt/ beta -catenin are related to the severity and prognosis of liver cancer. To sum up, Lnc- beta -Catm can promote the interaction of beta -catenin and EZH2, and then promote the methylation of beta -catenin, enhance the stability of beta -catenin, activate the Wnt/ beta -catenin signal pathway, and dry the liver cancer. We also found that the Yap1 signaling pathway was inhibited by the high expression of LncBrm in the liver cancer stem cells and in the cells which promoted the self renewing.LncBrm knockout, while the high expression of LncBrm promoted the activation of Yap1. Further studies found that LncBrm was interacting with Brm and inhibited the SWI/SNF complex of Brm type. Assembly, the assembly of Brg1 type SWI/SNF complexes that promote the assembly of.Brg1 type complexes can activate the expression of Yap1 by recruiting K1f4 to bind to the promoter region of Yap1. Yap1 signals promote the maintenance of self renewal of liver cancer stem cells. In conclusion, we identified the genes that play an important role in the stem cell of the tumor (C8orf4 and Zic2). And lncRNA (Lnc- beta -Catm and LncBrm) determined their effects on self-renewal of liver cancer stem cells and revealed their molecular mechanisms regulating self-renewal.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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