肝肿瘤线粒体功能障碍及抗氧化应激机制的探讨

Investigation of the mechanisms of mitochondrial dysfunction and antioxidative defense in hepatic tumor cells

目的 :探讨癌基因Ras诱导小鼠肝组织中线粒体功能的障碍及肝肿瘤细胞抗氧化应激反应的分子作用机制。方法 :采集H-ras12V转基因雄性小鼠的肝癌组织、癌旁组织以及正常健康雄性小鼠的肝组织(正常对照)。采用比色法定量检测肝组织中线粒体呼吸链复合物Ⅰ的活性;对线粒体呼吸链相关基因的转录组进行测序,并采用实时荧光定量PCR法检测组织中线粒体复合物关键酶NADH脱氢酶(辅酶)1α亚基12[NADH dehydrogenase(ubiquinone)1 alpha subcomplex 12,Ndufa12]、NADH脱氢酶(辅酶)铁硫蛋白6[NADH dehydrogenase(ubiquinone)Fe-S protein 6,Ndufs6]、泛醌细胞色素C还原酶结合蛋白(ubiquinol-cytochrome C reductase binding protein,Uqcrb)和细胞色素C氧化酶7a2(cytochrome C oxidase subunitⅦa polypeptide 2,Cox7a2)m RNA的表达水平以及Bcl-2和解偶联蛋白2(uncoupling protein2,Ucp2)m RNA的表达水平;免疫荧光法检测组织中活性氧(reactive oxygen species,ROS)的含量。结果 :与正常健康雄性小鼠肝组织相比,H-ras12V转基因雄性小鼠肝癌组织和癌旁组织中线粒体呼吸链复合物Ⅰ的活性均显著降低(F=14.26,P=0.003);转基因小鼠肝癌组织和癌旁组织中Ndufa12、Ndufs6、Uqcrb和Cox7a2 m RNA的表达水平均显著降低(F=18.55,P=0.001;F=7.06,P=0.014;F=12.81,P=0.002;F=10.51,P=0.004)。免疫荧光检测结果显示,与正常对照组和转基因小鼠肝癌组织相比,转基因小鼠癌旁组织中ROS的水平显著升高(F=31.35,P=0.001);Bcl-2和Ucp2m RNA表达水平均显著升高(F=20.29,P=0.002;F=15.51,P=0.001)。结论 :癌基因Ras的表达直接下调了肝组织线粒体呼吸链复合物关键酶的表达,进而导致线粒体功能障碍并上调细胞内ROS的水平。肝癌细胞通过促进Bcl-2和Ucp2的表达降低线粒体产生的ROS,进而促进肝癌的发生和发展。

Objective：To investigate the mechanisms of Ras oncogene-induced mitochondrial dysfunction and antioxidative defense in hepatic tumor cells.Methods：The hepatic tumor tissues and adjacent paracancerous tissues of H-ras12 V transgenic mice and the hepatic tissues of normal mice were collected.The activity of mitochondrial respiratory chain complex I was measured by quantitative colorimetric detection kit.The transcriptome of mitochondrial respiratory chain-related genes were sequenced.The mRNA expression levels of key enzymes of mitochondrial respiratory chain complex Ⅰ/Ⅲ/Ⅳ[NADH dehydrogenase（ubiquinone） 1 alpha subcomplex 12（Ndufa12）,NADH dehydrogenase（ubiquinone） Fe-S protein 6（Ndufs6）,ubiquinolcytochrome C reductase binding protein（Uqcrb） and cytochrome C oxidase subunit Ⅶa polypeptide 2（Cox7a2）],Bcl-2 and uncoupling protein 2（Ucp2） were detected by realtime fluorescence quantitative PCR.The reactive oxygen species（ROS） level was detected by immunofluorescence method.Results：Compared with the hepatic tissues of normal mice,in hepatic tumor tissues and adjacent paracancerous tissues of transgenic mice,the activity of mitochondrial respiratory chain complex I was significantly reduced（F = 14.26,P = 0.003）,and the mRNA expression levels of key enzymes of mitochondrial respiratory chain complex Ⅰ/Ⅲ/Ⅳ（Ndufal 2,Ndufs6,Uqcrb and Cox7a2） were significantly decreased（F = 1 8.55,P = 0.001;F = 7.06,P = 0.01 4;F = 12.81,P = 0.002;F = 10.51,P = 0.004）.Compared with the hepatic tissues of normal mice and hepatic tumor tissues of transgenic mice,the ROS level in adjacent paracancerous tissues was significantly increased（F = 31.35,P = 0.001）.Compared with the hepatic tissues of normal mice and hepatic tumor tissues of transgenic mice,the mRNA levels of Bcl-2 and Ucp2 in adjacent paracancerous tissues of transgenic mice were significantly increased（F =20.29,P = 0.002;F = 15.51,P = 0.001）.Conclusion：Ras oncogene directly reduces the transcriptional levels of the key enzymes of mitochondrial respiratory chain complex in hepatocytes which leads to mitochondrial dysfunction and high intracellular ROS level.In hepatic tumor cells,the elevated expression levels of Bcl-2 and Ucp2 contribute to the reduction of mitochondrial ROS level and the promotion of tumorigenesis and tumor progression.