转录因子YY1抑制FEN1基因表达及乳腺瘤细胞耐药性产生机理相关研究

发布时间：2018-02-13 10:25

本文关键词： 耐药性瓣状内切核酸酶1 YY1 丝裂霉素C 紫杉醇乳腺癌　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：耐药性是肿瘤治疗过程中一个非常大的挑战。大量的研究已经证实,重复的化学治疗会使细胞的DNA修复能力增强,从而导致耐药性的产生。但是,这种转变的精确的分子机理目前还不清楚。绝大多数化疗药物的药性主要依赖于诱导肿瘤细胞中DNA的损伤。然而,肿瘤细胞多重耐药性的产生是化学治疗中一个非常严峻的考验,也大大的限制了化疗药物的抗肿瘤效力。肿瘤细胞中药物转运和代谢途径的改变能够降低药物的抗肿瘤效力,但是抗药性的产生更与肿瘤细胞对DNA损伤承受能力的增加和DNA复制和修复能力的提升有关。瓣状核酸内切酶1(FEN1)在DNA复制和修复中发挥重要的作用。FEN1具有促进冈崎片段的成熟、长片段碱基切除修复、复制叉停顿的起始和稳定端粒的作用,因此能够维持基因组的稳定性,也曾一度被认为是一种肿瘤抑制因子。然而,越来越多的发现也说明,FEN1也参与肿瘤的发生过程。YY1是一个广泛分布的转录因子,能够调节多种基因的表达,在多种生物学过程如胚胎发育、分化、细胞增殖和肿瘤发生过程中多发挥重要作用。依据所处环境和所结合的蛋白因子的差异,YY1在调节基因表达时既可以作为激活因子也可以作为抑制因子。在本研究中我们发现YY1是FEN1基因表达的转录抑制因子。利用Match 1.0-public, TESS和TESEARCH等生物信息学软件对FEN1启动子序列进行预测,发现了包括NF-kB和YY1在内的大约200种转录因子。我们用纯化的YY1蛋白和一段包含预测的YYl结合位点的29bp探针进行了电泳迁移实验(EMSA),发现YY1能够与WT探针进行结合,形成稳定的YY1/DNA复合体。然而,如果将探针中保守位点的C替换成T,G替换成A,这种结合则不会存在。为了验证这种结合是否在细胞内发生,我们还进行了染色质免疫沉淀PCR (ChIP-PCR)实验,结果证明FEN1启动子能够被YY1特异性抗体拉下来,表明了YY1能够与FEN1启动子结合。我们在293T细胞中外源过表达了YY1并检测了FEN1蛋白的表达水平。我们发现,在293T细胞中随着外源过表达YY1质粒量的增加,内源FENl的蛋白水平呈现出逐渐降低的趋势。将克隆出的FEN1启动子插入到pGL4.10质粒中,来带动报告基因EGFP的表达。YY1过表达质粒和pGL4.10-FEN1 (promoter)-EGFP表达载体共转染到293T细胞中。分别利用qPCR和流式方法检测了EGFP的mRNA和蛋白荧光水平的表达。结果说明,在293T细胞中过表达YY1能够显著的降低EGFP基因的表达,也就是说YYl减弱了FEN1启动子的活性。对乳腺癌细胞MDA-MB-231进行丝裂霉素C (MMC)和紫杉醇处理,并通过qPCR和Western blotting来分析YY1和FEN1基因的表达变化。结果证明,MMC和紫杉醇处理后,YY1的mRNA水平降低了2倍以上,而FEN1的mRNA水平则升高了3到6倍。与此一致,在蛋白水平上,YY1降低了大约2倍,而FEN1则增加了2倍以上。此外,ChIP结果证明了MMC处理后YYl与FEN1启动子的结合降低了2倍。而且,我们还发现过表达YY1的细胞对MMC和紫杉醇的处理会更加敏感。用25种不同的DNA损伤试剂和化疗药物对13个类别的26种肿瘤细胞系进行了处理。Northern dot blotting结果说明,在乳腺癌细胞系MDA-MB-231和MDA-MB-4355中FEN1的表达在受到DNA损伤试剂如喜树碱、细胞松弛素D、MMC和伽马射线处理后会显著增加。临床研究结果表明,FEN1表达量较低的乳腺癌患者中超过80%的人可以活10年以上,然而在FEN1表达量高的乳腺癌患者中这个比例不超过55%。通过群体研究发现,FEN1高表达和YYl低表达的患者治愈率较低,表明这两个基因在乳腺癌患者的治疗过程中起相反的作用。这与我们利用细胞和药物处理得到的分子水平的研究结果一致,也表明了FEN1/YY1相互作用和调控机制具有一定的临床重要性。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

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