miR-462-731对草鱼线粒体功能的影响

Effects of miR-462-731 on the mitochondrial function of grass carp,Ctenopharyngodon idalla

硬骨鱼特有的miR-462与miR-731位于同一基因簇(简写为miR-462-731),在低氧环境中表达量会显著上调。为了进一步研究miR-462-731簇的作用,本研究以草鱼(Ctenopharyngodon idella)肝脏细胞为对象,分析miR-462与miR-731过表达对细胞线粒体生物学功能的影响。结果显示miR-462与miR-731过表达后三羧酸循环中关键基因mdh、ogdh、cs的mRNA表达水平以及ATP含量均显著降低,线粒体膜电位下降;细胞中活性氧(reactiveoxygen species,ROS)的含量显著增加,同时总抗氧化能力(totalantioxidantcapacity,T-AOC)及超氧化物歧化酶(superoxide dismutase,SOD)活性显著下降,而丙二醛(malondialdehyde,MDA)含量升高;电子显微镜观察发现miR-462与miR-731过表达后细胞线粒体结构损伤。由此推测,miR-462-731簇可通过影响细胞线粒体膜电位、代谢及氧化应激等参与线粒体活动,研究结果可为草鱼及其他硬骨鱼类低氧调节机制提供基础资料。

Oxygen is necessary for fish survival,and low oxygen levels affect the growth,reproduction,and survival of fish.miR-462 and miR-731 are located in the same gene cluster(miR-462-731),which is unique to teleost fish;the expression of this gene cluster is significantly upregulated in hypoxic environments.Mitochondria are the centers of energy metabolism and use oxygen for aerobic respiration to power the body.The function of miR-462-731 is speculated to be related to the mitochondria.To investigate whether the miR-462-731 cluster could regulate mitochondrial function in grass carp(Ctenopharyngodon idella),we analyzed the effects of miR-462 and miR-731 overexpression on the biological function of mitochondria in grass carp liver cells.The mRNA expression levels of key enzyme genes in the tricarboxylic acid cycle were detected by qRT-PCR after the overexpression of miR-462 and miR-731,and the expression of mdh,ogdh and cs significantly decreased.Simultaneously,the ATP content was significantly reduced.The JC-1 probe was used to detect the mitochondrial membrane potential of cells after transfection.The content of reactive oxygen species(ROS)in the cells increased significantly,whereas the total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)activity decreased significantly;however,the content of malondialdehyde(MDA)increased.Electron microscopy revealed that the overexpression of miR-462 and miR-731 led to mitochondrial structural damage.In conclusion,this study found that overexpression of miR-462-731 could reduce cellular energy metabolism and the antioxidant capacity of grass carp liver,destroy mitochondrial structure,and affect mitochondrial function.This study further improved the functional study of miR-462-731 and provided a theoretical basis for the hypoxic regulation mechanism and hypoxic tolerance of grass carp and other bony fishes.