内质网蛋白稳态在不同来源肿瘤及乳腺癌干细胞的调控中的作用机制研究

发布时间：2018-02-21 13:03

本文关键词： 未折叠蛋白反应内质网相关蛋白降解 p97 乳腺癌干细胞　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：目的探究内质网蛋白稳态调控中的未折叠蛋白反应(unfolded protein response, UPR)及内质网相关蛋白降解(endoplasmic reticulum associated degradation, ERAD)在不同来源肿瘤的发生发展及乳腺癌干细胞功能调控中的作用及其分子机制。方法免疫印记及免疫组织化学的方法分析在正常及应激条件下,不同来源肿瘤细胞及肿瘤组织中UPR及ERAD重要分子的蛋白表达及磷酸化激活水平；MTS细胞增殖实验及流式细胞术检测正常及应激条件下,UPR的三条信号通路及ERAD的小分子抑制剂对于不同来源肿瘤细胞增殖凋亡的影响；腺病毒感染目的基因过表达、RNA干扰、流式细胞术、体外培养及体外侵袭实验分析UPR及ERAD的小分子抑制剂及p97表达水平对于乳腺癌细胞增殖、凋亡、侵袭及乳腺癌干细胞体外功能的影响；裸鼠成瘤模型、流式细胞术及免疫组织化学的方法研究UPR及ERAD抑制剂对于乳腺癌干细胞体内功能的影响；基因芯片、实时定量PCR、免疫印记及RNA干扰的方法分析UPR及ERAD抑制剂处理乳腺癌细胞后,影响乳腺癌干细胞功能的分子机制。结果在正常及应激条件下,不同来源肿瘤细胞中UPR的重要分子的表达水平及其激活状态存在显著差异。在非应激条件下,UPR及ERAD的小分子抑制剂能够显著抑制不同来源肿瘤细胞的增殖,但仅有ERAD抑制剂能引起明显的细胞凋亡,而且UPR及ERAD的小分子抑制剂的敏感性存在差异。p97的蛋白表达水平与乳腺癌细胞的恶性程度、乳腺癌组织中Sox2的蛋白表达水平以及乳腺癌组织及细胞中CD44+/CD24肿瘤干细胞的比例呈正相关。改变乳腺癌细胞中p97的本底表达水平,会显著影响相应的乳腺癌干细胞的功能。p97抑制剂处理乳腺癌细胞后,可以显著降低其细胞中肿瘤干细胞的比例、体外成球能力及体外侵袭能力。p97抑制剂可以显著抑制由MDA-MB-231细胞所形成的裸鼠移植瘤的生长速度、其中乳腺癌干细胞的活性以及乳腺癌的复发。p97抑制剂Eer I与临床抗肿瘤化疗药物Doxorubicin及Bortezomib联合作用,可以显著抑制乳腺癌细胞的体外及体内的增殖、凋亡及乳腺癌干细胞的功能。最后,p97通过抑制C/EBP-δ及促进p53的表达,上调SKP2及Sox2的表达水平,最终维持了乳腺癌干细胞的功能。结论UPR三条信号通路及ERAD不同程度地影响普通乳腺癌细胞及乳腺癌干细胞的功能,p97由于在乳腺癌的生长及乳腺癌干细胞的功能中发挥的重要作用,有望成为抗肿瘤治疗的新靶点。
[Abstract]:Objective to investigate the role of unfolded protein response (UPRs) and endoplasmic reticulum associated degradation (ERAD) in the development of tumors from different sources and the regulation of the function of breast cancer stem cells in the steady-state regulation of endoplasmic reticulum (ER) proteins. Molecular mechanisms. Methods Immunoimprinting and immunohistochemical analysis under normal and stress conditions, Protein expression and phosphorylation Activation level of UPR and ERAD in tumor cells and tumor tissues from different sources; Detection of three signal Pathways and small score of ERAD in normal and stressed condition by flow Cytometry. The effect of subinhibitors on proliferation and apoptosis of tumor cells from different sources; Overexpression of target genes in adenovirus infection, flow cytometry, in vitro culture and invasion experiments were used to analyze the effects of small molecular inhibitors and p97 expression of UPR and ERAD on the proliferation and apoptosis of breast cancer cells. Invasion and function of breast cancer stem cells in vitro; tumorigenesis model of nude mice, flow cytometry and immunohistochemical methods to study the effects of UPR and ERAD inhibitors on the function of breast cancer stem cells in vivo; gene chip, The effects of UPR and ERAD inhibitors on the function of breast cancer stem cells were analyzed by real-time quantitative PCR, immunological imprinting and RNA interference. There were significant differences in the expression level and activation status of the important molecules of UPR in tumor cells from different sources. The small molecular inhibitors of UPR and ERAD could significantly inhibit the proliferation of tumor cells from different sources under non-stress conditions. However, only ERAD inhibitors could induce apoptosis, and the sensitivity of small molecular inhibitors of UPR and ERAD was different from the malignant degree of breast cancer cells. The expression level of Sox2 protein in breast cancer tissues and the proportion of CD44 / CD24 tumor stem cells in breast cancer tissues and cells were positively correlated. It has a significant effect on the function of the corresponding breast cancer stem cells. P97 inhibitor can significantly reduce the proportion of tumor stem cells in the breast cancer cells. P97 inhibitor could significantly inhibit the growth rate of xenografts formed by MDA-MB-231 cells in nude mice. The activity of breast cancer stem cells and the combination of breast cancer recurrence. P97 inhibitor Eer I with clinical antitumor chemotherapeutic drugs Doxorubicin and Bortezomib could significantly inhibit the proliferation of breast cancer cells in vitro and in vivo. Apoptosis and the function of breast cancer stem cells. Finally, p97 upregulated the expression of SKP2 and Sox2 by inhibiting the expression of C / EBP- 未 and promoting the expression of p53. Conclusion three signaling pathways of UPR and ERAD affect the function of breast cancer cells and breast cancer stem cells to some extent because of the growth of breast cancer and the function of breast cancer stem cells. To play an important role, It is expected to be a new target for anti-tumor therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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