本文關(guān)鍵詞： 耐藥性 瓣?duì)顑?nèi)切核酸酶1 YY1 絲裂霉素C 紫杉醇 乳腺癌　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：耐藥性是腫瘤治療過(guò)程中一個(gè)非常大的挑戰(zhàn)。大量的研究已經(jīng)證實(shí),重復(fù)的化學(xué)治療會(huì)使細(xì)胞的DNA修復(fù)能力增強(qiáng),從而導(dǎo)致耐藥性的產(chǎn)生。但是,這種轉(zhuǎn)變的精確的分子機(jī)理目前還不清楚。絕大多數(shù)化療藥物的藥性主要依賴于誘導(dǎo)腫瘤細(xì)胞中DNA的損傷。然而,腫瘤細(xì)胞多重耐藥性的產(chǎn)生是化學(xué)治療中一個(gè)非常嚴(yán)峻的考驗(yàn),也大大的限制了化療藥物的抗腫瘤效力。腫瘤細(xì)胞中藥物轉(zhuǎn)運(yùn)和代謝途徑的改變能夠降低藥物的抗腫瘤效力,但是抗藥性的產(chǎn)生更與腫瘤細(xì)胞對(duì)DNA損傷承受能力的增加和DNA復(fù)制和修復(fù)能力的提升有關(guān)。瓣?duì)詈怂醿?nèi)切酶1(FEN1)在DNA復(fù)制和修復(fù)中發(fā)揮重要的作用。FEN1具有促進(jìn)岡崎片段的成熟、長(zhǎng)片段堿基切除修復(fù)、復(fù)制叉停頓的起始和穩(wěn)定端粒的作用,因此能夠維持基因組的穩(wěn)定性,也曾一度被認(rèn)為是一種腫瘤抑制因子。然而,越來(lái)越多的發(fā)現(xiàn)也說(shuō)明,FEN1也參與腫瘤的發(fā)生過(guò)程。YY1是一個(gè)廣泛分布的轉(zhuǎn)錄因子,能夠調(diào)節(jié)多種基因的表達(dá),在多種生物學(xué)過(guò)程如胚胎發(fā)育、分化、細(xì)胞增殖和腫瘤發(fā)生過(guò)程中多發(fā)揮重要作用。依據(jù)所處環(huán)境和所結(jié)合的蛋白因子的差異,YY1在調(diào)節(jié)基因表達(dá)時(shí)既可以作為激活因子也可以作為抑制因子。在本研究中我們發(fā)現(xiàn)YY1是FEN1基因表達(dá)的轉(zhuǎn)錄抑制因子。利用Match 1.0-public, TESS和TESEARCH等生物信息學(xué)軟件對(duì)FEN1啟動(dòng)子序列進(jìn)行預(yù)測(cè),發(fā)現(xiàn)了包括NF-kB和YY1在內(nèi)的大約200種轉(zhuǎn)錄因子。我們用純化的YY1蛋白和一段包含預(yù)測(cè)的YYl結(jié)合位點(diǎn)的29bp探針進(jìn)行了電泳遷移實(shí)驗(yàn)(EMSA),發(fā)現(xiàn)YY1能夠與WT探針進(jìn)行結(jié)合,形成穩(wěn)定的YY1/DNA復(fù)合體。然而,如果將探針中保守位點(diǎn)的C替換成T,G替換成A,這種結(jié)合則不會(huì)存在。為了驗(yàn)證這種結(jié)合是否在細(xì)胞內(nèi)發(fā)生,我們還進(jìn)行了染色質(zhì)免疫沉淀PCR (ChIP-PCR)實(shí)驗(yàn),結(jié)果證明FEN1啟動(dòng)子能夠被YY1特異性抗體拉下來(lái),表明了YY1能夠與FEN1啟動(dòng)子結(jié)合。我們?cè)?93T細(xì)胞中外源過(guò)表達(dá)了YY1并檢測(cè)了FEN1蛋白的表達(dá)水平。我們發(fā)現(xiàn),在293T細(xì)胞中隨著外源過(guò)表達(dá)YY1質(zhì)粒量的增加,內(nèi)源FENl的蛋白水平呈現(xiàn)出逐漸降低的趨勢(shì)。將克隆出的FEN1啟動(dòng)子插入到pGL4.10質(zhì)粒中,來(lái)帶動(dòng)報(bào)告基因EGFP的表達(dá)。YY1過(guò)表達(dá)質(zhì)粒和pGL4.10-FEN1 (promoter)-EGFP表達(dá)載體共轉(zhuǎn)染到293T細(xì)胞中。分別利用qPCR和流式方法檢測(cè)了EGFP的mRNA和蛋白熒光水平的表達(dá)。結(jié)果說(shuō)明,在293T細(xì)胞中過(guò)表達(dá)YY1能夠顯著的降低EGFP基因的表達(dá),也就是說(shuō)YYl減弱了FEN1啟動(dòng)子的活性。對(duì)乳腺癌細(xì)胞MDA-MB-231進(jìn)行絲裂霉素C (MMC)和紫杉醇處理,并通過(guò)qPCR和Western blotting來(lái)分析YY1和FEN1基因的表達(dá)變化。結(jié)果證明,MMC和紫杉醇處理后,YY1的mRNA水平降低了2倍以上,而FEN1的mRNA水平則升高了3到6倍。與此一致,在蛋白水平上,YY1降低了大約2倍,而FEN1則增加了2倍以上。此外,ChIP結(jié)果證明了MMC處理后YYl與FEN1啟動(dòng)子的結(jié)合降低了2倍。而且,我們還發(fā)現(xiàn)過(guò)表達(dá)YY1的細(xì)胞對(duì)MMC和紫杉醇的處理會(huì)更加敏感。用25種不同的DNA損傷試劑和化療藥物對(duì)13個(gè)類別的26種腫瘤細(xì)胞系進(jìn)行了處理。Northern dot blotting結(jié)果說(shuō)明,在乳腺癌細(xì)胞系MDA-MB-231和MDA-MB-4355中FEN1的表達(dá)在受到DNA損傷試劑如喜樹(shù)堿、細(xì)胞松弛素D、MMC和伽馬射線處理后會(huì)顯著增加。臨床研究結(jié)果表明,FEN1表達(dá)量較低的乳腺癌患者中超過(guò)80%的人可以活10年以上,然而在FEN1表達(dá)量高的乳腺癌患者中這個(gè)比例不超過(guò)55%。通過(guò)群體研究發(fā)現(xiàn),FEN1高表達(dá)和YYl低表達(dá)的患者治愈率較低,表明這兩個(gè)基因在乳腺癌患者的治療過(guò)程中起相反的作用。這與我們利用細(xì)胞和藥物處理得到的分子水平的研究結(jié)果一致,也表明了FEN1/YY1相互作用和調(diào)控機(jī)制具有一定的臨床重要性。
[Abstract]:Drug resistance is a very big challenge in the process of cancer treatment. A number of studies have confirmed that chemical treatment will make repeated enhanced DNA repair capacity of cells, which leads to the emergence of drug resistance. However, the exact molecular mechanism of this change is unclear. Most chemotherapy drugs mainly depends on the induction of tumor cell in the DNA injury. However, multidrug resistance of cancer cells is a very severe test of chemical treatment, also greatly limits the effectiveness of chemotherapy resistant tumors. Tumor cells in drug transport and metabolism pathway changes can reduce the antitumor effect, but the resistance of tumor and more cells to DNA damage tolerance increased and DNA replication and repair ability. The flap endonuclease 1 (FEN1) play an important role of.FEN1 in DNA replication and repair To promote the maturation of Okazaki fragments, fragments of DNA repair, initiation and telomere of stalled replication forks, thus maintaining genome stability, also once considered to be a tumor suppressor. However, more and more discovery also shows that the process of.YY1 FEN1 is a transcription factor involved in tumor a widely distributed, to regulate the expression of various genes in the differentiation of many biological processes, such as embryonic development, cell proliferation and tumor play an important role in the process of difference. According to the environment and the combination of protein factors, YY1 can not only as the activator can also be used as inhibitors in regulating gene expression. In this study we found that YY1 is a transcription of FEN1 gene expression by Match inhibitor. 1.0-public, TESS and TESEARCH and other bioinformatics software on FEN1 promoter sequence Forecast, found about 200 kinds of transcription factors including NF-kB and YY1, we purified YY1 protein and contains a section of predicted YYl binding sites were 29BP probe (EMSA), electrophoretic migration experiment found that YY1 combined with the WT probe, the formation of a stable YY1/DNA complex. However, if the probe in the conservative site of C replaced T, G replaced A, this combination will not exist. In order to verify whether this combination occurs within the cell, we also conducted PCR chromatin immunoprecipitation (ChIP-PCR) experiments, results show that FEN1 promoter can be pulled down YY1 specific antibody that binds to YY1 the FEN1 promoter in 293T cells. We exogenous overexpression of YY1 and detected the expression level of FEN1 protein. We found that in 293T cells with exogenous overexpression of YY1 increased the amount of plasmid, flat water protein endogenous FENl gradually Decreased. The cloned FEN1 promoter was inserted into pGL4.10, to drive the expression of.YY1 gene EGFP expression plasmid and pGL4.10-FEN1 (promoter) -EGFP expression vectors were transfected into 293T cells respectively. Using qPCR and flow cytometry method to detect the expression of EGFP mRNA and protein levels. The results of fluorescence that overexpression of YY1 could significantly reduce the expression of EGFP gene in 293T cells, namely YYl attenuated FEN1 promoter activity of mitomycin C on breast cancer cell line MDA-MB-231 (MMC) and paclitaxel treatment, and through qPCR and Western blotting to analyze the expression of YY1 and FEN1 gene. The results showed that MMC, and treated with paclitaxel, YY1 mRNA levels were reduced by more than 2 times, while the FEN1 level of mRNA was increased by 3 to 6 times. Consistent with this, at the protein level, YY1 is about 2 times lower, while FEN1 increased more than 2 times. In addition, the ChIP results show that the combination of MMC YYl after treatment with FEN1 promoter was decreased 2 times. Moreover, we also found that overexpression of YY1 cells to MMC and paclitaxel will be more sensitive. The treatment of.Northern dot blotting showed that 26 kinds of tumor cell lines in 13 categories with 25 different DNA damage agents and chemotherapy drugs, in the FEN1 MDA-MB-231 and MDA-MB-4355 in breast cancer cell lines expressed in DNA damage agents such as camptothecin, cytochalasin D, MMC and gamma ray treatment will increase significantly. The clinical results showed that the expression of FEN1, more than 80% people were lower in patients with breast cancer can be live more than 10 years, but in the FEN1 high expression in breast cancer patients the ratio does not exceed 55%. by group study found that high expression of FEN1 and low expression of YYl in patients with low cure rate, suggesting that these two genes in breast cancer patients The treatment process has the opposite effect. This is consistent with our molecular level research results obtained from cell and drug treatment. It also indicates that FEN1/YY1 interaction and regulation mechanism has certain clinical importance.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.9

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;The ATF/CREB site is the key element for franscription of the human RNA methyransferase likel(RNMTL1)gene,a newly discovered 17p13.3 gene[J];Cell Research;2002年Z1期

2 Richard Y. ZHAO,Robert T. ELDER;Viral infections and cell cycle G2/M regulation[J];Cell Research;2005年03期

，

本文編號(hào)：1507940