Cai Quanxiang, Zhao Mengmeng, Liu Xiang, Wang Xiaochun, Nie Yao, Li Ping, Liu Tingyan, Ge Ruli, Han Fengchan
Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai 264003, Shandong, PR China; Department of Neurology, University Hospital of Binzhou Medical University, 661 Second Huanghe Road, Binzhou 256603, Shandong, PR China.
Key Laboratory for Genetic Hearing Disorders in Shandong, Binzhou Medical University, 346 Guanhai Road, Yantai 264003, Shandong, PR China; Department of Biochemistry and Molecular Biology, Binzhou Medical University, 346 Guanhai Road, Yantai 264003, Shandong, PR China.
Biochem Biophys Res Commun. 2017 Apr 1;485(2):388-394. doi: 10.1016/j.bbrc.2017.02.067. Epub 2017 Feb 16.
A/J mice are a mouse model of age-related hearing loss. It has been demonstrated that a mutation in gene of citrate synthase (CS) contributes to the early onset of hearing loss occurring at about one month of age. To understand the effects of a decreased CS activity that results from the mutation in Cs gene on hearing loss in A/J mice, human kidney cell line (293T) was transiently transfected with short hairpin RNA for Cs (shRNA-Cs) to reduce expression of CS. In comparison with those of cells transfected with a scrambled sequence (shRNA-NC), the oxygen consumption rate and adenosine trisphosphate (ATP) production level were decreased in 293T cells transfected with shRNA-Cs. Meanwhile, excessive superoxide production was induced as determined by mitochondrial superoxide formation assay (MitoSOX) and superoxide dismutase 2 (SOD2) detection. Moreover, the expression levels of BIP (binding immunoglobulin protein) and CHOP (CCAAT/enhancer-binding protein-homologous protein), markers of endoplasmic reticulum stress, were upregulated. Furthermore, apoptosis related molecule caspase-3 and the mitochondrial membrane potential were reduced. It is therefore concluded that downregulation of Cs expression in 293T cells leads to low level of ATP production, excessive superoxide formation and cell apoptosis, which implies a possible mechanism for hearing loss in A/J mice.
A/J小鼠是年龄相关性听力损失的一种小鼠模型。已经证明，柠檬酸合酶（CS）基因的突变促成了在约1月龄时发生的听力损失的早发。为了了解Cs基因突变导致的CS活性降低对A/J小鼠听力损失的影响，用针对Cs的短发夹RNA（shRNA-Cs）瞬时转染人肾细胞系（293T）以降低CS的表达。与用乱序序列（shRNA-NC）转染的细胞相比，用shRNA-Cs转染的293T细胞的耗氧率和三磷酸腺苷（ATP）产生水平降低。同时，通过线粒体超氧化物形成测定（MitoSOX）和超氧化物歧化酶2（SOD2）检测确定诱导了过量的超氧化物产生。此外，内质网应激标志物结合免疫球蛋白蛋白（BIP）和CCAAT/增强子结合蛋白同源蛋白（CHOP）的表达水平上调。此外，凋亡相关分子半胱天冬酶-3和线粒体膜电位降低。因此得出结论，293T细胞中Cs表达的下调导致ATP产生水平降低、过量的超氧化物形成和细胞凋亡，这暗示了A/J小鼠听力损失的一种可能机制。
