Cellular Signalling ( IF 4.8 ) Pub Date : 2020-09-12 , DOI: 10.1016/j.cellsig.2020.109779 Han Zhang 1 , Xiufang Zou 1 , Feng Liu 2
Noncoding RNAs are interweaved in pathological processes in myocardial ischemia (MI), such as long noncoding RNA (lncRNA) and microRNAs (miRNAs). The aim of this study was to figure out the role of Testis-specific transcript Y-linked 15 (TTTY15) and let-7i-5p in cell model of MI in cardiomyocytes. Hypoxia-induced cell injury was assessed by Cell counting kit 8 assay, flow cytometry, commercial kits and western blotting. As a result, hypoxia stress induced inhibition on cell proliferation, glucose uptake, and ATP production, and promotion on apoptosis, lactate dehydrogenase (LDH) release, and lactic acid production in human cardiomyocyte AC16 cells. During hypoxia injury, expression of TTTY15 and let-7i-5p was measured by real-time quantitative polymerase chain reaction, and TTTY15 was upregulated, accompanied with let-7i-5p downregulation. Functionally, either silencing TTTY15 or overexpressing let-7i-5p could attenuate hypoxia-induced apoptosis and mitochondrial energy metabolism dysfunction in AC16 cells. Moreover, there was an interaction between TTTY15 and let-7i-5p via target binding, as evidenced by dual-luciferase reporter assay and RNA immunoprecipitation assay. Knockdown of let-7i-5p could counteract the protective role of TTTY15 deletion in hypoxic AC16 cells. Meanwhile, toll-like receptor 3 (TLR3)/nuclear factor-kappa B (NF-κB) signaling was validated by western blotting. Expression of TLR3, tumor necrosis factor receptor-associated factor 6 (TRAF6) and phosphorylated p65 was promoted in hypoxic AC16 cells, which was abrogated by TTTY15 silencing along with let-7i-5p upregulation. Collectively, TTTY15 knockdown protects cardiomyocytes against hypoxia-induced apoptosis and mitochondrial energy metabolism dysfunction in vitro through let-7i-5p/TLR3/NF-κB pathway to suppress.
中文翻译:
沉默 TTTY15 通过 TTTY15/let-7i-5p 和 TLR3/NF-κB 通路减轻缺氧诱导的线粒体能量代谢功能障碍和心肌细胞凋亡。
非编码 RNA 交织在心肌缺血 (MI) 的病理过程中,例如长链非编码 RNA (lncRNA) 和微 RNA (miRNA)。本研究的目的是找出睾丸特异性转录物 Y-linked 15 (TTTY15) 和 let-7i-5p 在心肌细胞 MI 细胞模型中的作用。缺氧诱导的细胞损伤通过细胞计数试剂盒 8 测定、流式细胞术、商业试剂盒和蛋白质印迹法进行评估。因此,缺氧应激会抑制人心肌细胞 AC16 细胞的细胞增殖、葡萄糖摄取和 ATP 产生,并促进细胞凋亡、乳酸脱氢酶 (LDH) 释放和乳酸产生。缺氧损伤期间,实时定量聚合酶链反应检测TTTY15和let-7i-5p的表达,TTTY15上调,同时let-7i-5p下调。在功能上,沉默 TTTY15 或过表达 let-7i-5p 可以减轻缺氧诱导的 AC16 细胞凋亡和线粒体能量代谢功能障碍。此外,TTTY15 和 let-7i-5p 之间存在相互作用通过目标结合,如双荧光素酶报告基因测定和 RNA 免疫沉淀测定所证明的那样。敲低 let-7i-5p 可以抵消 TTTY15 缺失在缺氧 AC16 细胞中的保护作用。同时,通过蛋白质印迹验证了toll样受体3(TLR3)/核因子-κB(NF-κB)信号传导。TLR3、肿瘤坏死因子受体相关因子 6 (TRAF6) 和磷酸化 p65 的表达在缺氧 AC16 细胞中得到促进,这被 TTTY15 沉默和 let-7i-5p 上调所消除。总的来说,TTTY15 敲低通过 let-7i-5p/TLR3/NF-κB 通路抑制体外保护心肌细胞免受缺氧诱导的细胞凋亡和线粒体能量代谢功能障碍。