YY1转录因子对乳腺癌细胞自噬过程的表观遗传学调控作用及其机制研究

发布时间：2018-02-28 02:22

本文关键词： 乳腺癌自噬表观遗传学 YY1 MIR372 SQSTM1/p62 LC3　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：背景乳腺癌是全球范围内女性癌症发病率第一位的恶性肿瘤。自噬是细胞在应激条件下降解自身细胞质甚至细胞器以适应生存的关键生理反应,近年来的研究发现,细胞自噬水平紊乱与肿瘤发生发展密切相关。癌基因YYl转录因子(Yin Yang 1)已被报道在包括乳腺癌在内的恶性肿瘤中发挥重要作用,但其在乳腺肿瘤细胞自噬的相关调控机制尚未见报道。目的目前已有研究证实YY1涉及的致癌性机制可能有抑制细胞周期阻滞、抑制凋亡、抑制细胞分化等,但其在自噬中的调控作用却未见报道。我们希望通过一系列体内外实验来明确YY1基因是否通过影响自噬而在乳腺癌中发挥癌基因的生物学功能,探索其涉及到在肿瘤细胞自噬中的相应分子调控机制。方法用蛋白免疫印迹(Western Blotting)、免疫组化方法检测乳腺癌细胞水平和组织水平Y1、SQSTM1的表达情况及二者相关性；使用YY1 siRNA转染乳腺癌细胞MCF7和T47D,用蛋白酶体抑制剂和不同的自噬抑制剂处理后荧光显微镜观察、MTS等手段确认YY1对SQSTM1的调控；通过生物信息学软件分析可能靶向SQSTM1的miRNAs (microRNAs),实时荧光定量PCR (Real Time quantita-tive PCR)检测miRNA;运用染色质免疫共沉淀(Chromatin Immunoprecipitation)实验技术和双荧光素酶报告系统明确YYl对MIR372的表达调控,并通过甲基化特异性PCR (Methylation specific PCR)和甲基化DNA免疫共沉淀(Methylat-ed DNA Immunoprecipitation)验证YY1对MIR372启动子区域的甲基化水平的影响；构建SK-BR-3 YY1 shRNA稳定表达细胞系,将其与对照组细胞悬液接种于两组随机分组的裸鼠皮下建立裸鼠成瘤模型,取瘤称重,绘制生长曲线。结果本研究中首次证实YY1可以通过调控细胞自噬来影响肿瘤的发生发展。与正常乳腺上皮细胞和正常乳腺组织相比,YYl在乳腺癌细胞和原发乳腺癌组织中均呈高表达状态；同时,体内外实验均证实YY1和SQSTM1能促进乳腺癌细胞的生长,其蛋白水平表达呈正相关(p0.05)。YY1既不是通过影响SQSTM1的蛋白稳定性也不是通过影响其转录水平而影响SQSTM1的蛋白表达的。而MIR372则是唯一一个对SQSTM1的转录后水平进行调控并且其本身受YY1调控的miRNA。过表达MIR372可以表现出与YY1敲减后一致的细胞活力下降和自噬阻滞等一系列细胞表型和体内实验结果。而在YY1 siRNA处理敲减YY1基础上再敲减MIR372可以逆转YY1敲减所致细胞活力下降和自噬阻滞等一系列细胞表型以及SQSTM1蛋白的下调。EBSS诱导细胞自噬发生时,YY1和SQSTM1蛋白水平上调,MIR372水平下调,证实了YY1-MIR372-SQSTM1轴确实在肿瘤细胞的生理性自噬过程中产生调控作用,最终参与肿瘤的发生发展。结论YY1可以通过对乳腺癌细胞自噬的表观遗传学调控来影响肿瘤的发生发展,其作用机制是通过YY1-MIR372-SQSTM1轴实现对自噬的调控。
[Abstract]:Background Breast cancer is the first malignant tumor in women worldwide. Autophagy is the key physiological response of cells to degrade their cytoplasm or even cell organelles to adapt to survival under stress. The disorder of autophagy level is closely related to tumorigenesis and development. The oncogene YYl transcription factor Yin Yang 1 has been reported to play an important role in malignant tumors, including breast cancer. However, the regulation mechanism of autophagy in breast tumor cells has not been reported. Objective it has been confirmed that the carcinogenic mechanisms involved in YY1 may include inhibition of cell cycle arrest, inhibition of apoptosis, inhibition of cell differentiation, and so on. However, its role in autophagy has not been reported. We hope to find out whether YY1 gene plays a biological role in breast cancer by affecting autophagy through a series of experiments in vivo and in vitro. Methods Western blotting was used to detect the expression of Y1CS-SQSTM1 in breast cancer cells by immunohistochemistry. YY1 siRNA was used to transfect MCF7 and T47D into breast cancer cells. After treated with proteasome inhibitor and different autophagy inhibitor, fluorescence microscopy was used to confirm the regulation of YY1 on SQSTM1. SQSTM1 microRNA was analyzed by bioinformatics software, and real Time quantita-tive PCR was used to detect miRNAs. The expression regulation of YYl on MIR372 was determined by chromatin co-precipitation assay and double luciferase report system. The effect of YY1 on the methylation level of MIR372 promoter region was tested by methylation specific PCR method specific specific and methylated DNA immunoprecipitation, and a stable SK-BR-3 YY1 shRNA cell line was constructed. The model of tumorigenesis was established by inoculating the cell suspension with the control group into two groups of nude mice randomly divided into two groups, and the tumor was weighed. Results in this study, it is the first time that YY1 can influence the development of tumor by regulating autophagy. Compared with normal breast epithelial cells and normal breast tissues, YYl is present in breast cancer cells and primary breast cancer. High expression was found in all tissues. At the same time, in vivo and in vitro experiments confirmed that YY1 and SQSTM1 can promote the growth of breast cancer cells. The expression of MIR372 protein was positively correlated with the expression of SQSTM1 protein, and MIR372 was the only one that regulated the posttranscriptional level of SQSTM1 and had no effect on the protein stability of SQSTM1 or on the expression of SQSTM1 protein by affecting the transcriptional level of SQSTM1. The overexpression of MIR372, which is regulated by YY1, can show a series of cell phenotypes and in vivo experimental results, such as cell viability decline and autophagy arrest, which are consistent with YY1 knockout. The YY1 siRNA treatment of knockdown YY1 can also reduce MIR372. It can reverse a series of cell phenotypes such as cell viability decline and autophagy arrest induced by YY1 knockout and down-regulation of SQSTM1 protein. The levels of YY1 and SQSTM1 protein upregulate the down-regulation of MIR372 during the induction of autophagy. It is proved that the YY1-MIR372-SQSTM1 axis does play a regulatory role in the physiological autophagy of tumor cells, and eventually participate in the development of tumor. Conclusion YY1 can affect the development of tumor through epigenetic regulation of autophagy of breast cancer cells. The mechanism is that autophagy is regulated by YY1-MIR372-SQSTM1 axis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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