PDK1调节HepG2细胞能量代谢的机制研究

【作者】 刘颖；

【导师】 叶海燕；

【作者基本信息】 陕西师范大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 肿瘤发生机制的研究是生命科学领域的热点之一。肿瘤代谢的改变是肿瘤的主要特征之一,因此,研究肿瘤细胞的能量代谢机制对于研究肿瘤的发生机制及治疗具有重要的意义。丙酮酸脱氢酶(Pyruvate dehydrogenase complex,PDH)是一种连接糖酵解与氧化磷酸化的关键酶,其活性在多种肿瘤细胞中出现异常改变。有研究表明,P32与细胞自噬及氧化磷酸化等过程密切相关,通过与PDH的相互作用影响细胞能量代谢方式,将糖酵解转化为氧化磷酸化。由于丙酮酸脱氢酶激酶 1(Pyruvate dehydrogenase kinase 1,PDK1)是 PDH 的磷酸化激酶,也是糖酵解与氧化磷酸化的关键节点激酶,参与调节肿瘤细胞中Warburg效应的发生,因此,PDK1在肿瘤细胞能量代谢中发挥着重要的作用,但相关机制并不是十分清楚。为了探索PDK1参与肿瘤细胞能量代谢的机制,本课题将以肝癌细胞系HepG2为模型,通过以下几个方面进行相关实验研究,并取得了相应的实验结果。1.PDK1对HepG2细胞能量代谢的影响:使用PDK1的抑制剂二氯乙酸(Dichloroacetic acid,DCA)处理HepG2后,结果表明:细胞活力出现了一定程度的下降,HepG2细胞增殖减少;葡萄糖消耗量极显著升高,乳酸含量极显著降低;ATP含量显著升高,细胞产生的能量增加;G6PDH的活性及NADPH的含量均无明显变化。这些结果提示:PDK1的活性被抑制后,能够在一定程度上影响HepG2细胞的无氧酵解,提高细胞氧化呼吸水平。2.PDK1对HepG2细胞中P32和PDH水平及其在细胞内分布的影响:使用PDK1的抑制剂DCA处理HepG2后,结果表明.:HepG2细胞中PDH及其磷酸化pPDHA1(Ser293)的蛋白水平发生了变化,提示PDK1下游蛋白PDH被激活,HepG2细胞的代谢方式被部分逆转为氧化磷酸化。P32的蛋白表达水平发生变化,P32在线粒体中定位增加且与PDH的共定位增加。以上结果说明P32与PDH的相互作用与PDH的去磷酸化有关,其细胞定位的改变与PDK1的活性存在一定关系。3.PDK1对HepG2细胞线粒体形态的影响:使用PDK1的抑制剂DCA处理HepG2后,结果表明:HepG2细胞中线粒体融合蛋白(Mitofusin 2,Mfn2)的水平显著上升,线粒体分裂蛋白(Dynamin-related protein 1,Drp1)的水平极显著下降,表明PDK1通过调节PDH的活性控制氧化磷酸化的进程,还对线粒体的形态改变具有影响作用。提示PDK1对氧化磷酸化进程具有多重调节作用,可以通过影响线粒体分裂融合的平衡从而改变细胞氧化磷酸化水平。4.PDK1对HepG2细胞凋亡的影响:使用PDK1的抑制剂DCA处理HepG2后,结果表明:HepG2细胞中细胞凋亡相关蛋白Cleaved Caspase9,Cleaved Caspase3和Bax(Bcl-2 associated X protein)的水平均表现为先降低后升高的趋势,表明PDK1的活性被抑制后,HepG2细胞的凋亡增加。综上所述,PDK1作为PDH的激酶,可以调控细胞从无氧酵解到有氧呼吸过程的转换,抑制其活性可以提高细胞氧化呼吸水平;P32,PDK1与PDH存在相互作用,P32与PDH的相互作用与PDH的去磷酸化有关,P32细胞定位的改变与PDK1的活性有一定关系;结合三者定位情况推测三者共同参与了线粒体氧化磷酸化过程。即PDK1通过调节PDH的活性控制氧化磷酸化的进程,还通过对线粒体分裂融合的影响改变线粒体形态从而影响氧化磷酸化水平,PDK1对有氧呼吸进程具有多重调节作用;PDK1被抑制后细胞凋亡水平升高,说明线粒体内源性的细胞凋亡与氧化磷酸化和线粒体形态密切相关,肿瘤细胞的生长的逆转与线粒体分裂融合平衡有一定的关系。本课题研究结果为阐明PDK1调节肿瘤细胞能量代谢的机制研究奠定了重要基础,同时也为基于靶向肿瘤代谢的肿瘤治疗提供了新的理论依据。更多还原

【Abstract】 The research on the mechanism of tumorigenesis is one of the hot topics in the life science.The change of metabolism is one of the main characteristics of tumor.Thus,studying the mechanism of energy metabolism of tumor cells is of great significance for tumorigenesis mechanism and treatment research.As a key enzyme linking glycolysis and oxidative phosphorylation,pyruvate dehydrogenase complex(PDH)activity has abnormal changes in varieties of tumor cells.Studies have shown that P32 is closely related to cell autophagy,oxidative phosphorylation and other processes.The interaction between P32 and PDH influence the mode of energy metabolism-glycolysis will be converted to oxidative phosphorylation.As a phosphorylation kinase of PDH and key node kinase for glycolysis and oxidative phosphorylation,pyruvate dehydrogenase kinase 1(PDK1)also involved in the regulation of the Warburg effect in tumor cells.Therefore,PDK1 plays an important role in energy metabolism of tumor cells,but the exact mechanism isn’t very clear.In order to explore the mechanism of PDKI effect in the energy metabolism of tumor cells,the liver cancer cell line HepG2 will be used as a model for the following experimental research in this project.Results are as follows:1.The effect of PDK1 on the energy metabolism of HepG2 cells:Dichloroacetic acid(DCA)was used to inhibit PDK1 activity in HepG2 cells.The results showed that cell viability and proliferation decreased of HepG2 cell.Lactic acid content decreased significantly.Glucose consumption,ATP content and the energy produced by HepG2 cells increased significantly.There were no significant changes in G6PDH activity and NADPH content.It was shown that the anaerobic lysis of HepG2 cells can be affected by PDK1 inhibition to some extent and the level of oxidative respiration was increased.2.The effect of PDK1 on the level and intracellular distribution of P32 and PDH in HepG2 cells:After PDK1 was inhibited in HepG2 cells,the results showed that the level of PDH and pPDHA1(Ser293)changed,indicating that the downstream PDH of PDK1 was activated and the metabolism was partially reversed to oxidative phosphorylation in HepG2 cells.The level of P32 was changed.The location of P32 and co-localization with PDH were both increased in mitochondria after PDK1 inhibition.Results above showed that PDH dephosphorylation is related to the interaction between P32 and PDH.The change of cellular localization of P32 has a certain relationship with PDK1 activity.3.The effect of PDK1 on the morphology of mitochondria in HepG2 cells:Using DCA to inhibit PDK1 in HepG2 cells,the expression of mitochondrial fusion protein mitofusin 2(Mfn2)increased significantly while the mitochondrial division protein dynamin-related protein 1(Drp1)decreased significantly,indicating that PDK1 not_only control the process of oxidative phosphorylation through regulating the activity of PDH,but also has an effect on the morphological changes of mitochondria.PDK1 plays multiple roles-in regulating the process of oxidative phosphorylation and also changes the level of oxidative phosphorylation in cells through affecting the balance of mitochondrial division and fusion.4.The effect of PDK1 on the apoptosis of HepG2 cells:When PDK1 activity was inhibited by DCA in HepG2 cells,the results showed that the level of apoptosis-related proteins,such as Cleaved Caspase9,Cleaved Caspase3 and Bax(Bcl-2 associated X protein)showed a trend of decreasing first and then increasing,indicating that apoptosis was increased after PDK1 inhibition in HepG2 cells.In conclusion,as a kinase of PDH,PDK1 can regulate the conversion of anaerobic lysis to aerobic respiration in cells.The level of oxidative respiration can increase when PDK activity was inhibited in cells.P32,PDK and PDH interact with each other and the interaction of P32 and PDH is related to PDH dephosphorylation.The change of localization of P32 is related to the activity of PDK 1 in cells.Based on the co-localization of P32,PDK1 and PDH,it is speculated that the three participate in the mitochondrial oxidative phosphorylation.PDK1 controls the oxidative phosphorylation process by regulating the activity of PDH and the process of mitochondrial division and fusion to change the mitochondrial morphology.PDK1 has multiple regulatory effects on the aerobic respiration process.The apoptosis of cells was increased after PDK1 inhibition,indicating that oxidative phosphorylation and mitochondrial morphology tightly related to endogenous apoptosis of mitochondria.Reversal the growth of tumor cells has a certain relationship with the balance of mitochondrial division and fusion.Results of this project provide an important foundation for revealing the mechanism of PDK1 regulating energy metabolism of tumor cells,meanwhile provide a new theoretical basis for tumor treatment based on targeted tumor metabolism.更多还原