本文選題：肝癌 + 腫瘤干細胞��； 參考：《中國科學技術(shù)大學》2016年博士論文

【摘要】：原發(fā)性肝癌是全球五大常見癌癥之一,中國每年有超過46萬新發(fā)病例和42萬死亡患者,肝癌被稱為我國的“國病”,嚴重威脅著人們的健康水平。腫瘤干細胞學說認為在腫瘤組織中,只有極少量的致瘤性細胞亞群具有無限增生的潛能,具有自我更新和多向分化能力,產(chǎn)生異質(zhì)性腫瘤細胞,且對化療藥物有耐藥性。腫瘤干細胞在啟始腫瘤形成和生長中起著關(guān)鍵作用,目前的治療措施還無法針對腫瘤干細胞發(fā)揮作用,這可能是導致多種腫瘤復發(fā)、轉(zhuǎn)移和耐藥的主要原因。因此,若要治愈腫瘤,必須清除腫瘤中的干細胞。但是肝癌干細胞的自我更新機制仍需進一步闡明。我們前期研究發(fā)現(xiàn),CD133和CD13兩個分子的聯(lián)合應(yīng)用能夠分選出干性能力更強的肝癌干細胞。我們分選出肝癌干細胞(CD133+CD13+)和非干細胞(CD133-CD13-),做轉(zhuǎn)錄組芯片分析,鑒定了在腫瘤干細胞中起重要調(diào)節(jié)作用的基因(C8orf4和Zic2)與長鏈非編碼RNA (Lnc-β-Catm和LncBrm),并系統(tǒng)研究了它們對肝癌干細胞自我更新的調(diào)控作用及其分子機制。轉(zhuǎn)錄組分析和臨床樣本驗證均顯示C8orf4在肝癌和肝癌干細胞中低表達。C8orf4敲低之后,肝癌干細胞的小球形成能力和腫瘤起始能力削弱。進一步研究發(fā)現(xiàn),C8o1f4負調(diào)控Notch信號,并得到了臨床數(shù)據(jù)的支持。通過酵母雙雜交篩選,我們發(fā)現(xiàn)C8orf4與N2ICD (Notch2胞內(nèi)段)有相互作用,實驗證實C8orf4影響N2ICD的入核而N2ICD的入核對Notch2信號的活化至關(guān)重要。進一步研究表明,Notch2本身及其下游基因Nrarp、Hey1等在肝癌干細胞中也起著非常重要的作用,Notch2、Nrarp和Heyl敲低的細胞有較低的小球形成能力和腫瘤起始能力。還發(fā)現(xiàn)Notch靶基因的表達量和肝癌患者的臨床分期分級、臨床嚴重性和預(yù)后有關(guān)。Zic2在肝癌中高表達,在肝癌干細胞中表達量更高。研究表明Zic2敲除的細胞形成小球能力和腫瘤起始能力變?nèi)?Zic2高表達的細胞干性增強。檢測發(fā)現(xiàn),Zic2敲除的細胞中OCT4及其下游基因表達量變低,而Zic2高表達的細胞中OCT4表達量升高,證實Zic2能夠影響OCT4的表達。酵母雙雜交的結(jié)果顯示,Zic2能夠結(jié)合NURF復合物,而N JRF復合物對于OCT4啟動子區(qū)的染色體開放和啟動轉(zhuǎn)錄至關(guān)重要�？傊�,Zic2通過招募NURF復合物,啟動OCT4的表達,繼而參與肝癌干細胞自我更新的維持。長鏈非編碼RNA (lncRNA)是一類重要的調(diào)節(jié)分子,在基因表達和其它多種生物學過程中發(fā)揮重要作用。我們發(fā)現(xiàn),Lnc-β-Catm分布于細胞核中,調(diào)節(jié)肝癌干細胞的小球形成能力和腫瘤起始能力。Lnc-β-Catm敲低的細胞干性削弱,而高表達Lnc-β-Catm的細胞干性增強。進一步研究發(fā)現(xiàn),Lnc-β-Catm影響Wnt/β-cateni n信號通路。Lnc-β-Catm能夠結(jié)合β-catenin和EZH2, EZH2能夠甲基化β-catenin,甲基化的P-catenin穩(wěn)定性增加。通過臨床樣本的檢測,發(fā)現(xiàn)Lnc-β-Catm的表達量和Wnt/β-catenin的活性與肝癌的嚴重程度和預(yù)后相關(guān)。綜上所述,Lnc-β-Catm能夠促進β-catenin和EZH2的相互作用,進而促進β-catenin的甲基化,增強了β-catenin的穩(wěn)定性,從而活化了Wnt/β-catenin信號通路,在肝癌干細胞自我更新維持中發(fā)揮重要作用。我們還發(fā)現(xiàn),LncBrm在肝癌干細胞中高表達并促進其自我更新。LncBrm敲低的細胞中,Yap1信號通路受到抑制,而LncBrm高表達則促進Yap1的活化。進一步研究發(fā)現(xiàn),LncBrm與Brm相互作用,并抑制Brm類型的SWI/SNF復合物的組裝,促進Brg1類型的SWI/SNF復合物的組裝。Brg1類型的復合物能夠通過招募K1f4結(jié)合到Y(jié)ap1的啟動子區(qū)域,啟動Yap1的表達,Yap1信號在促進了肝癌干細胞自我更新的維持�？傊�,我們鑒定了在腫瘤干細胞中起重要調(diào)節(jié)作用的基因(C8orf4 和 Zic2)與lncRNA(Lnc-β-Catm和LncBrm),確定了它們對肝癌干細胞自我更新的影響及其揭示了其調(diào)控自我更新的分子機制。
[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.
【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7

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