MCUR1介导线粒体钙稳态重塑调控ROS产生促进肝癌生长的作用机制研究

发布时间：2019-06-09 18:26

【摘要】：线粒体钙稳态对线粒体功能及细胞的影响至关重要,线粒体钙维持胞浆钙离子浓度动态平衡,是线粒体呼吸功能的重要调节因素,钙离子失衡导致细胞凋亡。线粒体钙稳态重塑是多种疾病发生的主要原因,但这一改变是否在肿瘤恶性进展中发挥关键作用尚无明确报道。我们的研究团队首次发现:新近报道的线粒体钙离子摄取关键调节分子MCUR1,促进肝癌细胞粒体钙稳态摄取,其表达水平在肝癌组织细胞中异常升高,MCUR1高表达能够通过加速细胞增殖抑制线粒体依赖的内源性细胞凋亡而促进了肝癌细胞的生长能力,并且与肝癌病人的不良预后相关。文献报道,线粒体的钙离子环境是调控细胞活性氧ROS生成的关键因素,而活性氧ROS作为胞内重要的第二信使,能够激活包括与肿瘤细胞增殖,凋亡,转移与肿瘤细胞的血管生成相关的多种关键信号通路,最终导致恶性肿瘤发生。我们进一步研究发现MCUR1介导的线粒体钙稳态重塑导致肝癌细胞活性氧ROS水平上调,这一改变与其下游P53降解,AKT/MDM2信号通路活化密切相关。基于此,我们提出假说:MCUR1上调引发线粒体钙稳态重塑,通过调控胞内第二信使ROS生成影响其下游akt/mdm2介导的p53降解信号通路,最终促进肝癌的恶性进展。【目的】本课题以肝癌为研究对象,选择线粒体钙离子摄取关键调节分子mcur1,明确mcur1介导的线粒体钙稳态重塑是否参与对肝癌生长的调控,并探索mcur1介导的线粒体钙稳态重塑调控肝癌生长的作用机制。【方法】1.利用免疫组化实验分析mcur1以及p53在原发性肝癌临床标本的表达及相关性。2.利用mts法测定细胞生长曲线、克隆形成实验、annexin-v凋亡检测、流式细胞周期检测、edu细胞增殖等实验方法,分析mcur1对肝癌细胞增殖凋亡及生长的影响。3.利用裸鼠皮下荷瘤实验,分析mcur1在体内对肝癌生长的影响。4.利用rhod-2,fluo-4活细胞染色,分析mcur1对肝癌细胞线粒体基础钙离子水平以及线粒体对胞浆钙的缓冲作用的影响。5.利用dcf及mitosox染料对细胞ros及线粒体ros进行染色,分析mcur1对肝癌细胞线粒体ros生成的影响。6.利用westernblot检测凋亡相关分子,以及增殖相关分子的表达改变,分析mcur1介导的线粒体钙稳态重塑促进肝癌细胞的生长的作用机制。【结果】第一部分:通过对原发性肝癌临床标本进行分析,发现mcur1在肝癌组织中表达上调,与肝癌不良预后相关。通过采用mts法测定细胞生长曲线、克隆形成实验、annexin-v凋亡检测、流式细胞周期检测、edu细胞增殖等实验方法,发现肝癌细胞中mcur1通过抑制细胞凋亡并且加速细胞增殖而促进肝癌细胞的生长。通过进行裸鼠皮下荷瘤实验,证实mcur1通过抑制肝癌凋亡,加速肝癌增殖,而促进肝癌的生长。第二部分:通过rhod-2,fluo-4染色检测线粒体钙以及胞浆钙,发现:mcur1能够促进线粒体钙离子摄入,提升线粒体基础钙离子浓度,并且在应急状态下线粒体对胞浆钙的缓冲力。通过对线粒体单向转运体mcu进行干预处理,发现mcur1依赖于mcu促进肝癌细胞线粒体钙的摄取。第三部分:通过对细胞ROS以及线粒体ROS的检测,发现MCUR1能够促进肝癌细胞线粒体ROS生成。通过线粒体的ROS清除剂Mito-TEMPO对细胞中线粒体ROS进行清除,以及线粒体钙清除载体PV-Mito对线粒体内的钙离子进行清除,发现MCUR1介导的线粒体钙离子稳态重塑通过促进肝癌细胞线粒体ROS生成,而抑制细胞凋亡促进细胞增殖。第四部分:通过检测凋亡相关分子,增殖相关分子,P53,AKT及MDM2磷酸化的检测,证实:MCUR1介导的线粒体钙稳态重塑通过ROS/AKT/MDM2途径使P53失活促进肝癌细胞生长。【结论】本课题研究发现:MCUR1介导肝癌线粒体钙离子摄入增强,重塑的线粒体钙稳态影响ROS的生成,通过ROS/AKT/MDM2途径使P53失活,调控肝癌的增殖凋亡,最终促进肿瘤进一步恶化。
[Abstract]:The steady state of mitochondrial calcium is critical to the function of the mitochondria and the effect of the cell, and the dynamic balance of the calcium ion concentration in the mitochondria is an important regulation factor of the mitochondrial respiratory function, and the imbalance of the calcium ions leads to the apoptosis of the cells. The steady-state remodeling of mitochondria is the main cause of various diseases, but it is not clear that this change plays a key role in the development of the malignant tumor. Our team first found that the newly-reported mitochondrial calcium ion uptake key regulatory molecule, MCU1, promotes the steady uptake of calcium and calcium in the liver cancer cell, and the expression level of the mitochondrial calcium ion is abnormally elevated in the liver cell of the liver cancer, The high expression of MCUR1 can promote the growth ability of the liver cancer cells by accelerating the cell proliferation and inhibiting the mitochondria-dependent endogenous cell apoptosis, and is related to the poor prognosis of the liver cancer patients. The literature reports that the calcium ion environment of mitochondria is a key factor to regulate the production of reactive oxygen ROS, and reactive oxygen ROS, as a second messenger in the cell, can activate various key signal pathways involved in the proliferation, apoptosis, metastasis and angiogenesis of tumor cells, Resulting in the occurrence of a malignant tumor. We further study that MCUR1-mediated steady-state remodeling of mitochondrial calcium results in an up-regulation of reactive oxygen ROS level in liver cancer cells, which is closely related to the downstream P53 degradation, AKT/ MDM2 signaling pathway activation. Based on this, we put forward the hypothesis that the MUR1 upregulates the steady-state remodeling of mitochondrial calcium, and by regulating the second messenger ROS in the cell, the downstream akt/ mdm2-mediated p53 degradation signal pathway is generated, and the malignant progression of the liver cancer is finally promoted. [Objective] To study whether the steady-state remodeling of mitochondrial calcium mediated by mcur1 is involved in the regulation of the growth of the liver cancer, and to explore the mechanism of the role of mcur1-mediated steady-state remodeling of the mitochondria in the control of the growth of the liver cancer. [Method] 1. The expression and correlation of mcur1 and p53 in the clinical specimens of primary liver cancer were analyzed by immunohistochemistry. The effects of mcur1 on the proliferation and apoptosis and growth of liver cancer cells were analyzed by using the method of mts to determine cell growth curve, clone formation experiment, annexin-v apoptosis detection, flow cytometry cycle detection and edu cell proliferation. The effect of mcur1 on the growth of liver cancer in vivo was analyzed using the nude mice's subcutaneous tumor-bearing experiment. The effect of mcur1 on the calcium ion level of the mitochondria of the liver cancer cells and the effect of the mitochondria on the calcium in the cytoplasm of the liver cancer cells was analyzed by using the r2005-2 and fluo-4 living cells. The effects of mcur1 on the generation of mitochondria in liver cancer cells were analyzed by using dcf and mittox dyes to dye the cells and the mitochondria. By using the western blot to detect the apoptosis-related molecules and the change of the expression of the related molecules, the mechanism of the role of mcur1-mediated steady-state remodeling of the mitochondrial calcium to promote the growth of the liver cancer cells was analyzed. [Results] The first part: Through the analysis of the clinical specimen of primary liver cancer, it was found that the expression of mcur1 in the liver cancer tissue was up-regulated and related to the bad prognosis of the liver cancer. By using the method of mts to determine the cell growth curve, the clone formation experiment, the annexin-v apoptosis test, the flow cytometry cycle detection, the edu cell proliferation, and the like, the mcur1 in the liver cancer cell is found to promote the growth of the liver cancer cell by inhibiting the cell apoptosis and accelerating the cell proliferation. By carrying out the subcutaneous tumor-bearing experiment of the nude mouse, the mcur1 is confirmed to promote the growth of the liver cancer by inhibiting the apoptosis of the liver cancer and accelerating the proliferation of the liver cancer. In the second part, the mitochondrial calcium and the cytosolic calcium were detected by rF-2 and fluo-4 staining, and it was found that mcur1 can promote the calcium ion uptake of mitochondria, increase the calcium ion concentration of the mitochondria, and reduce the buffering force of the mitochondria to the cytosolic calcium in the emergency state. It was found that mcur1 was dependent on the uptake of mitochondrial calcium in liver cancer cells. The third part: through the detection of ROS and the mitochondrial ROS, it is found that the MCU1 can promote the generation of the mitochondrial ROS in the liver cancer cells. The mitochondrial ROS in the cells was removed by the mitochondrial ROS scavenger, Mito-TEMPO, and the mitochondrial calcium removal vector PV-Mito was removed from the mitochondria, and the MUR1-mediated steady-state remodeling of the mitochondrial calcium ions was found by promoting the generation of mitochondrial ROS in the liver cancer cells, And the cell apoptosis is inhibited and the cell proliferation is promoted. The fourth part: Through the detection of the apoptosis-related molecules, the proliferation-related molecules, the P53, AKT and MDM2 phosphorylation, it was confirmed that the MUR1-mediated steady-state remodeling of the mitochondria promoted the growth of the liver cancer cells through the ROS/ AKT/ MDM2 pathway. [Conclusion] The study found that MCUR1 mediates the increase of mitochondrial calcium ion uptake in liver cancer, and the remodeling of the mitochondrial calcium homeostasis affects the production of ROS, and by means of the ROS/ AKT/ MDM2 pathway, the P53 is inactivated, the proliferation and apoptosis of the liver cancer are regulated, and the further deterioration of the tumor is promoted.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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