發(fā)布時(shí)間：2018-11-12 12:01

【摘要】：乳腺癌是世界范圍內(nèi)女性最常見(jiàn)的惡性腫瘤。雖然對(duì)乳腺癌主要采取手術(shù)、放療并輔以化療的聯(lián)合治療方案獲得很大成功,但仍有40%的乳腺癌患者在治療后出現(xiàn)腫瘤復(fù)發(fā)和癌細(xì)胞轉(zhuǎn)移,是導(dǎo)致乳腺癌患者死亡的主要原因。最近的研究證明,乳腺癌干細(xì)胞(Breast cancer stem cells, BCSCs)是乳腺癌復(fù)發(fā)、轉(zhuǎn)移和抵抗治療的關(guān)鍵原因。研究證明上皮一間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)是腫瘤細(xì)胞發(fā)生侵襲轉(zhuǎn)移的必經(jīng)途徑。另一方面,文獻(xiàn)報(bào)道發(fā)生EMT的乳腺癌細(xì)胞呈現(xiàn)BCSCs的特征。本論文的目的是分別從BCSCs和EMT兩個(gè)方面研究乳腺癌復(fù)發(fā)與轉(zhuǎn)移的機(jī)制,尋找開(kāi)發(fā)抗乳腺癌復(fù)發(fā)與轉(zhuǎn)移的潛在分子藥靶。大量研究證明,炎癥反應(yīng)可能是促進(jìn)BCSCs形成與維持的關(guān)鍵原因,但炎癥與BCSCs生成與維持的分子機(jī)制仍有待闡明。TRB3 (Tribbles homolog 3)是假性蛋白激酶成員之一,最近的研究證明,TRB3不僅與乳腺癌的不良預(yù)后呈正相關(guān),而且促進(jìn)腫瘤的侵襲和轉(zhuǎn)移。本實(shí)驗(yàn)室前期工作發(fā)現(xiàn),炎癥和應(yīng)激反應(yīng)增加炎癥損傷組織和腫瘤組織的TRB3表達(dá)。因此我們提出假說(shuō),TRB3介導(dǎo)炎癥反應(yīng)引起的BCSCs生成及維持。檢測(cè)乳腺癌組織芯片我們發(fā)現(xiàn)乳腺癌組織中炎癥因子IL6、TRB3和轉(zhuǎn)錄因子Sox2的表達(dá)呈正相關(guān)。利用磁珠分選技術(shù),我們發(fā)現(xiàn)由IL6誘導(dǎo)的CD44+/CD24- BCSCs田胞群TRB3含量遠(yuǎn)高于CD44-/CD24+分化細(xì)胞群。敲除內(nèi)源性TRB3的乳腺癌細(xì)胞株,BCSCs的形成能力明顯降低；過(guò)表達(dá)TRB3的乳腺癌細(xì)胞株BCSCs的形成和體內(nèi)成瘤的能力顯著升高。說(shuō)明TRB3促進(jìn)IL6誘導(dǎo)的BCSCs生成及維持。通過(guò)檢測(cè)TRB3對(duì)促干細(xì)胞蛋白Sox2、Oct4、Nanog表達(dá)及Sox2啟動(dòng)子活性的影響,我們發(fā)現(xiàn)TRB3促進(jìn)Sox2的基因轉(zhuǎn)錄。利用siRNA技術(shù)篩選調(diào)節(jié)Sox2的關(guān)鍵轉(zhuǎn)錄因子,我們發(fā)現(xiàn)轉(zhuǎn)錄因子Fox01介導(dǎo)TRB3促進(jìn)Sox2的基因轉(zhuǎn)錄。本研究發(fā)現(xiàn)TRB3抑制FoxO1的蛋白酶體降解途徑,同時(shí)增加Fox01基因轉(zhuǎn)錄,最終促進(jìn)Sox2的基因轉(zhuǎn)錄。免疫共沉淀的結(jié)果證明TRB3通過(guò)與Aktl相互作用,抑制Aktl與FoxO1結(jié)合,從而抑制FoxO1的蛋白酶體降解途徑。大量FoxO1在細(xì)胞核內(nèi)結(jié)合Sox2啟動(dòng)子并促進(jìn)Sox2基因轉(zhuǎn)錄。令我們興奮的發(fā)現(xiàn)是Sox2能與FoxOl啟動(dòng)子結(jié)合并促進(jìn)Fox01基因轉(zhuǎn)錄,從而在Sox2與Fox01之間形成正反饋環(huán)路,最終促進(jìn)BCSCs的形成。綜上所述,炎癥因子IL6增加TRB3的表達(dá),增加的TRB3通過(guò)抑制Aktl-FoxO1軸的活性,促進(jìn)Sox2的基因轉(zhuǎn)錄。增加的Sox2也促進(jìn)Fox01的基因轉(zhuǎn)錄,形成正反饋調(diào)節(jié)環(huán)路,進(jìn)而促進(jìn)BCSCs的形成。本研究首次證明TRB3促進(jìn)IL6介導(dǎo)的BCSCs形成及維持；闡明了炎癥因子IL6促進(jìn)BCSCs形成的分子機(jī)制。TRB3與Akt1相互作用可能是抑制BCSCs維持、抵抗乳腺癌復(fù)發(fā)及轉(zhuǎn)移的潛在靶點(diǎn)。目前研究證明,BCL6 (B-cell CLL/lymphoma6)在乳腺癌組織中高表達(dá),并促進(jìn)乳腺癌細(xì)胞的侵襲與轉(zhuǎn)移。但具體的分子機(jī)制有待闡明。EMT是腫瘤細(xì)胞發(fā)生侵襲與轉(zhuǎn)移的關(guān)鍵步驟,在乳腺癌的發(fā)生與發(fā)展中發(fā)揮重要作用。因此我們提出假說(shuō),BCL6通過(guò)誘導(dǎo)EMT的發(fā)生促進(jìn)乳腺癌的侵襲與轉(zhuǎn)移。利用Transwell、 Anchorage-independent growth和細(xì)胞免疫熒光實(shí)驗(yàn),我們發(fā)現(xiàn)BCL6通過(guò)誘導(dǎo)EMT的發(fā)生促進(jìn)乳腺癌細(xì)胞的生長(zhǎng)、侵襲與轉(zhuǎn)移能力。BCL6通過(guò)抑制上皮細(xì)胞標(biāo)志蛋白E-cadherin的基因轉(zhuǎn)錄,促進(jìn)EMT的發(fā)生。利用siRNA技術(shù)篩選抑制E-cadherin基因轉(zhuǎn)錄的轉(zhuǎn)錄因子,我們發(fā)現(xiàn)ZEB1介導(dǎo)BCL6誘導(dǎo)EMT和侵襲與轉(zhuǎn)移的能力。利用染色質(zhì)免疫共沉淀技術(shù),我們發(fā)現(xiàn)BCL6促進(jìn)ZEB1結(jié)合E-cadherin啟動(dòng)子,抑制E-cadherin的基因轉(zhuǎn)錄。同時(shí)我們發(fā)現(xiàn)BCL6能與ZEB1啟動(dòng)子結(jié)合,促進(jìn)ZEB1基因轉(zhuǎn)錄。說(shuō)明BCL6通過(guò)促進(jìn)ZEB1基因轉(zhuǎn)錄,抑制E-cadherin的基因轉(zhuǎn)錄。并且BCL6抑制劑79-6抑制BCL6在乳腺癌細(xì)胞中的以上功能。綜上所述,BCL6促進(jìn)ZEB1基因轉(zhuǎn)錄,ZEB1對(duì)E-cadherin的轉(zhuǎn)錄抑制作用增加,最終促進(jìn)乳腺癌細(xì)胞發(fā)生EMT。本研究首次證明BCL6促進(jìn)乳腺癌細(xì)胞發(fā)生EMT；闡明了BCL6促進(jìn)乳腺癌發(fā)生侵襲與轉(zhuǎn)移的分子機(jī)制。BCL6有可能成為抵抗乳腺癌侵襲與轉(zhuǎn)移的潛在靶標(biāo)。
[Abstract]:Breast cancer is the most common malignant tumor in the world. Although surgery, radiotherapy and chemotherapy-assisted combined therapy for breast cancer have been successful, 40% of patients with breast cancer have a recurrence of tumor and metastasis of cancer cells after treatment, leading to the death of breast cancer patients. Recent studies have shown that breast cancer stem cells (BCSCs) are the key causes of the recurrence, metastasis and resistance of breast cancer. It is proved that Epithelium-Mesenchymal transition (EMT) is a necessary route for the invasion and metastasis of tumor cells. On the other hand, the literature reports that the breast cancer cells of EMT have the characteristics of BCSCs. The purpose of this thesis is to study the mechanism of recurrence and metastasis of breast cancer from two aspects of BCSCs and EMT. A large number of studies have shown that the inflammatory response may be a key cause of the formation and maintenance of BCSCs, but the molecular mechanism of the formation and maintenance of inflammation and BCSCs remains to be clarified. TRB3 (Tribbles homolog 3) is one of the members of the pseudo-protein kinase. Recent studies have shown that TRB3 is not only positively related to the poor prognosis of breast cancer, but also promotes the invasion and metastasis of the tumor. The preliminary work of this lab found that inflammation and stress response increased the expression of TRB3 in the tissue and the tumor tissue of the inflammation. So we put forward the hypothesis that TRB3 mediated the formation and maintenance of BCSCs caused by inflammatory response. The expression of the inflammatory factors IL6, TRB3 and the transcription factor Sox2 in the breast cancer tissue was positively correlated with the detection of the breast cancer tissue chip. Using magnetic bead sorting, we found that the content of TRB3 in the cell group of CD44 +/ CD24-BCSCs induced by IL6 was much higher than that of CD44-/ CD24 + differentiated cells. The formation of BCSCs and the ability of BCSCs in breast cancer cell lines expressing TRB3 were significantly higher than that of BCSCs. Explain that TRB3 facilitates the generation and maintenance of BCSCs induced by IL6. TRB3 was found to promote the gene transcription of Sox2 by detecting the effect of TRB3 on the expression of stem cell protein Sox2, Oct4, Nanog and the activity of Sox2 promoter. The key transcription factor of Sox2 was screened by the siRNA technique, and we found that the transcription factor Fox01 mediates the transcription of the gene of the Sox2 by the transcription factor Fox01. In this study, the proteasome degradation pathway of FoxO1 was inhibited by TRB3, and the transcription of Fox01 gene was increased, and the gene transcription of Sox2 was promoted. The results of the immunoprecipitation demonstrated that TRB3 inhibited the proteasome degradation pathway of FoxO1 by interacting with Aktl to inhibit the binding of Aktl to FoxO1. A large amount of FoxO1 binds to the Sox2 promoter in the nucleus and promotes the transcription of the Sox2 gene. It is found that the Sox2 can be combined with the FoxO1 promoter and promote the transcription of the Fox01 gene, so that a positive feedback loop is formed between the Sox2 and the Fox01, and finally the formation of the BCSCs is promoted. In conclusion, the inflammatory factor IL6 increases the expression of TRB3, and the increased TRB3 promotes the gene transcription of the Sox2 by inhibiting the activity of the Aktl-FoxO1 axis. The increased Sox2 also promotes the gene transcription of Fox01, forming a positive feedback regulation loop, and further promoting the formation of BCSCs. This study first demonstrated that TRB3 promotes the formation and maintenance of BCSCs mediated by IL6, and illustrates the molecular mechanism for promoting the formation of BCSCs by the inflammatory factor IL6. The interaction between TRB3 and Akt1 may be a potential target to inhibit the maintenance of BCSCs and to resist the recurrence and metastasis of breast cancer. The present study has shown that BCL6 (B-cell CLL/ lymphalo6) is highly expressed in breast cancer tissues and promotes the invasion and metastasis of breast cancer cells. but specific molecular mechanisms need to be clarified. EMT is a key step in the invasion and metastasis of tumor cells, and plays an important role in the occurrence and development of breast cancer. Therefore, we put forward the hypothesis that BCL6 promotes the invasion and metastasis of breast cancer by inducing EMT. We found that BCL6 could promote the growth, invasion and transfer of breast cancer cells by induction of EMT. BCL6 promotes the occurrence of EMT by inhibiting the gene transcription of the epithelial cell marker protein E-cadherin. Using siRNA technology to screen the transcription factors that inhibit the transcription of E-cadherin gene, we find that ZEB1 mediates the ability of BCL6 to induce EMT and invasion and metastasis. Using the chromatin immunoprecipitation technique, we found that BCL6 promoted the expression of ZEB1 in the E-cadherin promoter and inhibited the gene transcription of E-cadherin. At the same time, we found that the BCL6 can be combined with the ZEB1 promoter to promote the transcription of the ZEB1 gene. This suggests that BCL6 can inhibit the gene transcription of E-cadherin by promoting the transcription of the ZEB1 gene. and the BCL6 inhibitor 79-6 inhibits the above function of the BCL6 in the breast cancer cell. In conclusion, the BCL6 promotes the transcription of the ZEB1 gene, and the transcription inhibition effect of the ZEB1 on E-cadherin is increased, and finally, the EMT of the breast cancer cell is promoted. In this study, BCL6 was used to promote the development of EMT in breast cancer cells, and the molecular mechanism of BCL6 to promote the invasion and metastasis of breast cancer was explained. BCL6 is a potential target to resist the invasion and metastasis of breast cancer.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R737.9

【共引文獻(xiàn)】

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8 汪健;SDF-1/CXCR4軸在肺癌轉(zhuǎn)移中作用與機(jī)制的研究[D];第四軍醫(yī)大學(xué);2009年

9 楊守華;上皮性卵巢腫瘤相關(guān)微淋巴管內(nèi)皮細(xì)胞的體外培養(yǎng)及其特性研究[D];華中科技大學(xué);2009年

10 孫仁虎;CCL21/CCR7在結(jié)腸癌侵襲與轉(zhuǎn)移過(guò)程中生物學(xué)意義的研究[D];華中科技大學(xué);2009年

相關(guān)碩士學(xué)位論文 前10條

1 王琛;SDF-1、CXCR4在食管癌中的表達(dá)及對(duì)預(yù)后的影響[D];鄭州大學(xué);2010年

2 理國(guó)富;CXCR4和VEGF在人腦星形細(xì)胞瘤中的表達(dá)及其與血管生成的關(guān)系[D];鄭州大學(xué);2010年

3 李宣朋;膠質(zhì)瘤的CXCR4表達(dá)及瓊脂糖下體外侵襲模型的建立[D];昆明醫(yī)學(xué)院;2011年

4 張麗娟;CXCR4在結(jié)直腸癌組織中的表達(dá)以及對(duì)SW480細(xì)胞增殖能力的影響[D];蘭州大學(xué);2011年

5 楊永德;局部進(jìn)展期乳腺癌趨化因子受體CXCR4的表達(dá)與新輔助化療療效的相關(guān)性及預(yù)后研究[D];重慶醫(yī)科大學(xué);2011年

6 錢(qián)伶凌;人參皂甙Rg3對(duì)乳腺癌細(xì)胞系的抑制作用[D];浙江工業(yè)大學(xué);2010年

7 陳濤;SDF-1/CXCR4軸在小細(xì)胞肺癌侵襲轉(zhuǎn)移中的作用及機(jī)制研究[D];蘇州大學(xué);2011年

8 宋萌萌;趨化因子CXCL12/CXCR4在子宮內(nèi)膜癌組織中的表達(dá)特征及意義[D];青島大學(xué);2011年

9 安松林;趨化因子受體CXCR4在乳腺癌中的表達(dá)及意義[D];天津醫(yī)科大學(xué);2006年

10 張春輝;CXCR4、VEGF和MMP-9在乳腺癌中的表達(dá)及其臨床意義[D];蘇州大學(xué);2006年

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