PPARδ基因增加乳腺癌细胞在恶劣代谢环境中生存率的研究

发布时间：2018-02-07 14:47

本文关键词： 乳腺癌过氧化物酶体增殖物激活受体delta(PPARδ) 葡萄糖剥夺氧化应激 AKT PPARδ拮抗剂　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：背景与目的:致死性乳腺癌细胞其新陈代谢具有杀伤正常细胞而使自身存活的特点。此特点可引起其对化疗及免疫治疗的抵抗,而最终成为难以治愈的癌症。针对这种让乳腺癌细胞可以在恶劣生存环境下存活的机制,目前我们发现一种更好的治疗靶点,也许可以成为新的特异性靶向治疗,从而改善治疗效果。细胞核受体——过氧化物酶体增殖物激活受体delta(PPARδ)是一种可以让癌细胞在恶劣生存环境下茁壮成长的关键调节因子。PPARδ是核受体PPAR家族的一员,这其中还包括PPARα和PPARγ,PPARδ可调控脂肪细胞中的脂肪储备以及肝脏和肌肉中的脂肪酸氧化。PPARδ的表达是普遍存在的,但当配体缺失或结合辅阻遏物(如NCo R1或去乙酰组蛋白)时可抑制其表达。它可以被高浓度游离脂肪酸、具有生物活性的脂质以及人工合成激动剂(GW501516、GW0742)激活。随着配体结合,PPARδ构象发生变化,继而介导基因转录(如PPARD本身、ANGPTL4、抗氧化基因CAT等),这些也是PPARδ被激活的标志。PPARδ可通过降低氧化应激反应和防止细胞分裂来增加骨骼肌耐力及阻止造血干细胞消耗。在这种氧化应激的条件下,细胞可在逐渐恶化的环境中保持相对长时间的存活。如果在癌细胞中PPARδ可以像在肌肉或干细胞中那样被活化,那么它也许也会在这种应激的代谢环境中继续生长,这也是浸润性癌症的特点之一。我们已经证实了,在白血病细胞中,当糖酵解被抑制时,PPARδm RNA和蛋白表达上调。本文我们将研究乳腺癌在不利条件下(固体肿瘤微环境)PPARδ的作用,为其在今后临床应用打下基础。方法:为了明确PPARδ在乳腺癌中的作用,我们利用逆转录病毒和慢病毒感染,建立PPARδ过表达及敲除乳腺癌细胞模型,并给予相应激动剂与拮抗剂进行干预,观察乳腺癌细胞在低糖、缺氧等不利生存条件下细胞的增殖及生存能力。通过免疫印迹方法和实时荧光定量PCR测定PPARδ相关蛋白和基因的表达。通过7AAD与DCFH染色流式细胞术测定细胞凋亡及ROS水平。通过Transwell细胞侵袭实验观察体外细胞迁徙能力。最后通过小鼠乳腺癌模型的建立,观察乳腺癌细胞在体内的生长与转移能力。结果:我们通过一组公共数据获知乳腺癌患者的生存率与PPARδ的表达相关,随后我们建立了大鼠乳腺癌细胞模型,发现癌细胞的快速生长(体外与体内)也与PPARδ的高表达有关。我们又将PPARD基因转染入人乳腺癌细胞株MCF-7与SKB-R3,发现转染了PPARδ的人乳腺癌细胞株具有快速迁徙(体外)与增加肿瘤转移(体内)的特点,而且PPARδ可以在营养匮乏的培养基中继续良好生长。之后我们又将人乳腺癌细胞株置于低浓度葡萄糖或其他内质网应激环境下(如缺氧),通过7AAD染色流式细胞术分析,发现转染了PPARD基因的人乳腺癌细胞株可以更好的存活。之后我们应用糖皮质激素或人工合成激动剂上调PPARδ表达同样可以使野生型MCF-7细胞在低糖环境下继续存活。DCFH染色流式细胞分析提示低糖环境下,高表达PPARδ细胞具有较高的抗氧化应激能力,从而提高乳腺癌细胞的生存率,随后给予对照组细胞过氧化氢酶处理也可增加其生存率。蛋白免疫印迹试验表明在低糖环境中高表达PPARδ细胞具有更高表达的迟发性AKT的磷酸化(葡萄糖剥夺后延长生存信号反应),进而延长乳腺癌细胞生存时间,同时给予胰岛素或干扰素α于对照组细胞增加AKT水平同样可增加其生存率,相反给予AKT抑制剂IV于PPARD高表达细胞可降低其生存率。最后应用人工合成PPARδ抑制剂,可以逆转这种PPARδ所产生的生存优势,这个逆转结果还可被其激动剂还原。结论:1、PPARδ可以通过降低氧化应激反应调节乳腺癌细胞在恶劣代谢环境中的生存率;2、PPARδ可以通过增强生存信号(p-AKT)调节乳腺癌细胞在恶劣代谢环境中的生存率;3、小分子抑制剂可以逆转PPARδ在乳腺癌中的保护作用;4、PPARδ可作为一种新的治疗靶点,为乳腺癌的治疗提供新的思路及方法。
[Abstract]:Background and purpose: fatal breast cancer cells and normal cells with the The new supersedes the old. the characteristics of their own survival. This characteristic can cause the resistance to chemotherapy and immunotherapy, and eventually become difficult to cure cancer. The mechanism of this that breast cancer cells can survive in the harsh environment, we found that the current therapeutic target a little better, may become the new targeting therapy, so as to improve the therapeutic effect. Nuclear receptor, peroxisome proliferator activated receptor delta (PPAR delta) is one of the key can make cancer cells thrive in the harsh environment under the regulatory factor.PPAR delta is a member of the nuclear receptor of the PPAR family, the including PPAR and PPAR expression of alpha gamma, PPAR Delta.PPAR delta can be fatty acid oxidation regulation in fat cells and fat reserves in liver and muscle is common, but When combined with ligand deletion or corepressor (such as NCo or R1 deacetylates histones) can inhibit its expression. It can be a high concentration of free fatty acids, bioactive lipids and synthetic agonists (GW501516, GW0742). With the activation of ligand binding, PPAR delta conformation occurs changes, then mediated transcription of genes (such as PPARD itself, ANGPTL4, CAT, antioxidant genes) are also activated PPAR Delta.PPAR delta sign can reduce oxidative stress and prevent cell division to increase muscle endurance and prevent the consumption of hematopoietic stem cells. In this oxidative stress conditions, cells can maintain a relatively long time survival in the deteriorating environment. If the cancer cells PPAR 8 can be like in muscle or stem cells that are activated, then perhaps it will continue to grow in the metabolism of environmental stress, which is characteristic of invasive cancer One. We have confirmed that in leukemia cells, when glycolysis was inhibited, up-regulated expression of PPAR delta m RNA and protein. In this paper, we will study on breast cancer under adverse conditions (solid tumor microenvironment) PPAR delta function, as in the future clinical application. Methods: in order to effect clear PPAR 8 in breast cancer, we use retroviral and lentiviral infection, the establishment of PPAR 8 overexpression and knockout breast cancer cell model, and give the corresponding agonist and antagonist intervention, observed in low sugar breast cancer cell proliferation and viability of cells in hypoxia disadvantageous living conditions. The determination of the expression of PPAR related proteins and genes by Western blot and real-time quantitative PCR. Flow cytometry was used to detect cell apoptosis by 7AAD staining and the level of ROS and DCFH. Through the Transwell cell invasion in vitro cell migration ability. Finally, through the establishment of a mouse model of breast cancer, breast cancer cell growth and metastasis in vivo. Results: our ability through the expression of survival rate and PPAR 8 a common set of data that patients with breast cancer related, then we established a rat model of breast cancer cells, the rapid growth of cancer cells in vitro and (in the high expression of PPAR) and 8. We will PPARD gene transfected into human breast cancer cell lines MCF-7 and SKB-R3 showed that the transfected PPAR 8 human breast cancer cell line with rapid migration (in vitro) and tumor metastasis (in vivo) characteristics, and can continue to grow well in PPAR Delta medium the lack of culture nutrition. Then we will be human breast cancer cell lines in a low concentration of glucose or other endoplasmic reticulum stress environment (such as hypoxia), flow cytometry analysis by 7AAD staining, found PPARD transfected human breast Survival can be better cancer cells. Then we use of corticosteroids or synthetic agonists up-regulated PPAR delta also makes the expression of wild type MCF-7 cells in low sugar environment to survive.DCFH staining and flow cytometry analysis indicated that a low sugar environment, high expression ability of PPAR delta cells have higher oxidative stress, so as to improve the survival the rate of breast cancer cells, then the cells of control group were given treatment with catalase can increase the survival rate. Western blotting test showed that the high expression of PPAR delta cells with higher expression of delayed phosphorylation of AKT in low sugar environment (the survival signal response after glucose deprivation) and prolong the survival of cancer cells, breast time, while giving insulin or interferon alpha increased AKT levels in the control group cells also can increase the survival rate, instead given AKT inhibitor IV in PPARD high expression cell can be reduced The survival rate. Finally, the application of synthetic inhibitor of PPAR Delta, Delta PPAR can reverse the resulting survival advantage, the results can be reversed by the agonist reduction. Conclusion: 1, PPAR 8 can lower survival rates of breast cancer by regulating cell metabolism in harsh environment in response to oxidative stress; 2, PPAR can. By enhancing the survival signal (p-AKT) in breast cancer cell survival rate in severe metabolic environment; 3, small molecule inhibitors can protect the reversal effect of PPAR 8 in breast cancer; 4, PPAR 8 can be used as a new therapeutic target, to provide new ideas and methods for the treatment of breast cancer.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

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