低氧诱导因子1a在前列腺癌细胞中脂代谢和自噬的调控作用研究

发布时间：2018-03-13 05:12

本文选题：前列腺癌　切入点：低氧诱导因子　出处：《武汉大学》2015年博士论文　论文类型：学位论文

【摘要】：目的：前列腺癌是男性泌尿生殖系统中的恶性肿瘤。在世界范围内,前列腺癌已经成为在男性中最为高发的恶性肿瘤之一,在美国每七个男性就有一个会被诊断为前列腺癌。而低氧是肿瘤的重要特征之一,在前列腺癌中低氧诱导因子(HIF1a)的高表达与前列腺癌的恶性程度和去势抵抗有着十分密切的联系。此外,近年的研究发现在肿瘤中脂代谢高表达和自噬的异常会促进肿瘤的发生发展。本研究着重探讨低氧诱导因子(HIFla)对前列腺癌细胞脂代谢和自噬的作用及其调控机制。方法：通过实时定量-聚合酶链反应(Real Time- Polymerase Chain Reaction, RT-PCR)与免疫印迹法(Western blot)比较不同前列腺癌细胞中HIF1a与脂肪合成基因的表达。在此基础上通过低氧环境(1%O2)培养与转染HIF1a突变质粒,分析脂肪合成基因的表达。并且通过荧光素酶报告基因与染色质免疫沉淀方法探究该变化的分子机制,’通过裸鼠实验证实HIF1a可以促进脂合成基因的表达。另一方面,成功利用低氧环境诱导前列腺癌细胞发生自噬后,通过实时定量-聚合酶链反应(Real Time-Polymerase Chain Reaction, RT-PCR)与免疫印迹法(Western blot)检测自噬相关基因的表达,并通过荧光素酶报告基因与染色质免疫沉淀方法详细解答内深层次的分子机制。结果：前列腺癌小鼠模型TRAMP的前列腺组织中,HIF1a高表达。在前列腺癌细胞中,缺氧诱导因子(HIF1a)与脂肪合成基因的表达存在一定相关性。前列腺癌细胞在缺氧条件下以及转染HIF1a突变质粒的条件下均可促进脂肪合成基因的表达。缺氧诱导因子(HIF1a)可以结合到SREBP-1c的启动子区域促进其转录。与对照组相比(1.000± 0.095,1.000±0.042), HIFla可以显著升高SREBP-1c的mRNA(2.992±0.159)与蛋白质(2.337±0.113)的表达水平。在裸鼠成瘤的结果中,由于HIF1a的过表达也呈现出脂合成基因SREBP-1c,ACC1和FASN都上调的结果,并伴随肿瘤体积的增大。此外,在前列腺癌细胞中,低氧的环境可以诱导前列腺癌发生自噬,自噬相关基因在RNA水平和蛋白质水平均显著升高。并且HIFla可以通过促进自噬关键元件ATG5的转录达到促进细胞自噬的效果。结论：缺氧的环境中,HIFla会通过激活SREBP-1c转录,从而促进前列腺癌细胞的从头脂肪合成；激活ATG5转录,促进前列腺癌的自噬。
[Abstract]:Objective: prostate cancer is a malignant tumor in the male genitourinary system. In the world, prostate cancer has become one of the most common malignant tumors in men. One in seven men in the United States is diagnosed with prostate cancer, and hypoxia is an important feature of the tumor. The high expression of hypoxia inducible factor (HIF1a) in prostate cancer is closely related to the malignancy and castration resistance of prostate cancer. Recent studies have found that hyperlipidemia and abnormal autophagy may promote the development of tumor. This study focuses on the effect of hypoxia inducible factor HIFla-induced lipid metabolism and autophagy in prostate cancer cells and its regulatory mechanism. Methods: real time Polymerase Chain reaction (RT-PCRR) was used to compare the expression of HIF1a and fat synthesis genes in different prostate cancer cells by real time Polymerase Chain reaction (RT-PCRR). To analyze the expression of fat synthesis gene, and to explore the molecular mechanism of this change by luciferase reporter gene and chromatin immunoprecipitation. After successfully induced autophagy in prostate cancer cells by hypoxia, real Time-Polymerase Chain reaction (RT-PCR) and Western blotting were used to detect the expression of autophagy related genes. The deep molecular mechanism was explained in detail by luciferase reporter gene and chromatin immunoprecipitation. Results: HIF1a was overexpressed in prostate tissue of prostate cancer mouse model TRAMP. Hypoxia inducible factor (HIF1a) is related to the expression of adipose synthase gene. Prostate cancer cells can promote the expression of adipose synthesis gene under hypoxia and transfection of HIF1a mutant plasmid. Hypoxia inducible factor (HIF1a) can promote the expression of adipose synthase gene. HIF1a) can bind to the promoter region of SREBP-1c to promote its transcription. Compared with the control group, HIFla can significantly increase the expression levels of mRNA(2.992 卤0.159) and 2.337 卤0.113) of SREBP-1c. In addition, in prostate cancer cells, hypoxia can induce autophagy in prostate cancer cells due to the up-regulation of lipid synthesis gene SREBP-1cACC1 and FASN, and the increase of tumor volume. Autophagy related genes were significantly increased at both RNA and protein levels, and HIFla could promote autophagy by promoting transcription of ATG5, a key component of autophagy. Conclusion: HIFla can activate SREBP-1c transcription in hypoxic environment. This promotes the ab initio fat synthesis of prostate cancer cells, activates ATG5 transcription, and promotes autophagy of prostate cancer.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

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