SLC25A10抑制肝癌凋亡的作用及机制研究

发布时间：2018-07-04 15:48

本文选题：SLC25A10 + 肝癌　；参考：《大连医科大学》2017年硕士论文

【摘要】：世界范围内,肝癌作为一种常见肿瘤,其发病率和死亡率均居高不下。有数据显示,全球50%的新发和死亡肝癌病例均来自中国。世界卫生组织预计,如不采取紧急行动,2015年至2030年间中国将有约1000万人死于肝癌。目前,手术仍作为肝癌治疗的主要手段,但手术后患者的五年生存率仍仅为15%-40%。因此,寻找新的治疗方法和新的治疗靶点是目前肝癌临床的当务之急,也是本课题的研究重点。人溶质载体蛋白第25号家族中A亚家族的第10位成员SLC25A10作为一种线粒体膜上的转运蛋白,主要将苹果酸和琥珀酸等二羧酸盐从线粒体内转运至线粒体外,以交换磷酸盐、硫酸盐及硫代硫酸盐,从而为糖异生和尿素合成等过程提供底物,进而维持TCA循环过程中的中间产物在线粒体内外的分布和稳态。近年来,有多篇文献报道转运蛋白SLC25A10在多种肿瘤中表达水平较高,但其在肝癌中的表达水平如何,鲜有文献报道,且其分子机制尚不明确。根据前期实验结果证实,转运蛋白SLC25A10在肝癌中有较高水平的表达且能够抑制肝癌凋亡的发生,本课题旨在进一步明确SLC25A10抑制肝癌凋亡发生的分子机制,找到治疗肝癌的新的潜在的靶点,更好的对肝癌进行靶向治疗。通过对多例临床肝癌组织和癌旁组织进行免疫组化分析,结果显示,SLC25A10于肝癌组织中高表达。同时,通过Realtime-PCR和Western Blot,在mRNA和蛋白水平对以上结果进行了进一步验证,得到了同样的结果。然后,通过慢病毒感染的方式,分别对SLC25A10表达水平较低的肝癌细胞SMMC-7721进行SLC25A10的稳定高表达,对SLC25A10表达水平较高的肝癌细胞HepG2进行SLC25A10的稳定低表达。通过细胞计数、流式凋亡检测以及对凋亡发生的标志性蛋白Cleaved Caspase-3蛋白水平的变化进行检测,高表达SLC25A10之后明显抑制肝癌细胞SMMC-7721凋亡的发生,并且在用经典凋亡诱导药物Etoposide处理之后,可以更明显的抑制凋亡的发生。而在肝癌细胞HepG2中低表达SLC25A10之后进行上述实验,获得了与高表达组正好相反的结果。在随后的体内实验中,裸鼠皮下成瘤同样证实了高表达SLC25A10之后能够抑制肝癌凋亡的发生。这其中的分子机制在于,高表达SLC25A10之后可以抑制线粒体途径的凋亡发生,即减少线粒体内cytochrome c的释放,抑制cytochrome c和Apaf-1结合后与Caspase-9形成凋亡复合物,从而抑制下游的凋亡效应蛋白如Caspase-3的激活,最终抑制凋亡的发生。综上所述,在肝癌中高表达的SLC25A10是通过抑制线粒体凋亡通路参与到肿瘤的发生发展过程中的,SLC25A10可能作为治疗肝癌的潜在靶点,实现对肝癌的靶向治疗。
[Abstract]:Worldwide, liver cancer as a common tumor, its morbidity and mortality are high. Data show that 50% of the world's new and death cases of liver cancer are from China. The World Health Organization estimates that without urgent action, about 10 million people in China will die from liver cancer between 2015 and 2030. At present, surgery is still the main method for the treatment of liver cancer, but the five-year survival rate of postoperative patients is only 15-40. Therefore, finding new treatment methods and new therapeutic targets is an urgent task in the clinical practice of liver cancer, and is also the research focus of this topic. SLC25A10, the tenth member of the A subfamily of Human Solute Carrier protein No. 25, is a transporter of mitochondrial membrane which transports dicarboxylate such as malic acid and succinic acid from the mitochondria to exchange phosphate. Sulfate and thiosulfate provide substrates for the processes of glycosylation and urea synthesis and thus maintain the distribution and homeostasis of intermediate products in and out of mitochondria during TCA cycle. In recent years, there have been many reports about the high expression of SLC25A10 in many kinds of tumors, but the expression level of SLC25A10 in HCC is rare, and its molecular mechanism is not clear. According to the previous experimental results, the transporter SLC25A10 is highly expressed in HCC and can inhibit the apoptosis of HCC. The purpose of this study is to further clarify the molecular mechanism of SLC25A10 inhibiting apoptosis in HCC. Find new potential targets for liver cancer and better target therapy for liver cancer. The expression of SLC25A10 was found to be high in clinical liver cancer tissues and paracancerous tissues by immunohistochemical analysis. At the same time, the results were further verified by Realtime-PCR and Western blot at mRNA and protein levels, and the same results were obtained. Then, the stable high expression of SLC25A10 in SMMC-7721 cells with lower SLC25A10 expression and the stable and low expression of SLC25A10 in HepG2 cells with higher SLC25A10 expression were detected by lentivirus infection. By cell count, flow cytometry and the change of Caspase-3 protein level, the expression of SLC25A10 significantly inhibited the apoptosis of SMMC-7721 cells, and the expression of SLC25A10 significantly inhibited the apoptosis of SMMC-7721 cells, and the expression of SLC25A10 significantly inhibited the apoptosis of SMMC-7721 cells. After treated with Etoposide, a classical apoptosis inducing drug, apoptosis was inhibited more obviously. The results were opposite to those in the high expression group after the low expression of SLC25A10 in HepG2 cells. In subsequent experiments in vivo, subcutaneous tumorigenesis in nude mice also confirmed that high expression of SLC25A10 could inhibit apoptosis of HCC. The molecular mechanism is that the overexpression of SLC25A10 can inhibit the apoptosis of mitochondrial pathway, that is, decrease the release of cytochrome c in mitochondria, inhibit the binding of cytochrome c and Apaf-1 to Caspase-9 and form apoptotic complex. Thus inhibiting the activation of downstream apoptosis-effector proteins such as Caspase-3 and ultimately inhibiting the occurrence of apoptosis. In conclusion, the high expression of SLC25A10 in HCC may be a potential target for the treatment of HCC by inhibiting the mitochondrial apoptotic pathway and participating in the process of tumor development.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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