PNPase调控线粒体microRNA对线粒体DNA的保护作用.docVIP

PNPase调控线粒体microRNA对线粒体DNA的保护作用 李力力，游 扬，周 源，凌贤龙 （400037重庆，第三军医大学新桥医院消化内科目的PNPase表达对线粒体microRNA（MitomiRs）作用。方法SK-Hep1、HepG2U2OS为研究对象，带绿色荧光（GFP）PNPaseshRNA 慢病毒转染细胞estern blot 方法检测PNPase细胞线粒体microRNA芯片检测下调PNPase前后线粒体microRNA（MitomiRs）种类的变化；Q-PCR检测mtDNA损伤ELISA方法检测8-羟基脱氧鸟苷（8-OHdG）。结果SK-Hep1、HepG2和U2OS细胞空载体病毒及PNPase shRNA慢病毒转染后1224h可见明显的绿色荧光。当MOI（转染复数）=20时，种细胞的慢病毒转染效率达80％以上。连续观察2周，转染效率仍维持在70％~80％。estern blot检测结果显示PNPase组PNPase蛋白表达低于正常组及组microRNA芯片显示PNPase shRNA后目的细胞MitomiRs发生明显变化，miR-30c-2-3p、miR-494、miR-1273g-3p、miR-4443表达上调miR-324-3p、miR-574-5p、miR-371b-5p、miR-6068、miR-21-5p表达下调。Q-PCR显示PNPase组mtDNA损伤减少ELISA结果显示PNPase组8-OHdG含量下降。结论抑制肿瘤细胞PNPase表达MitomiRs表达谱发生变化，肿瘤细胞mtDNA损伤MitomiRs可能参与了mtDNA复制的调控。 PNPase ；线粒体 ；MitomiRs ；氧化损伤Regulation of MitomiRs by PNPase inhibition prevents mitochondrial DNA from damage Li Lili, You Yang, Zhou Yuan, Ling Xianlong (Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China) [Abstract] Objective To investigate the regulation of mitochondrial microRNAs (MitomiRs) by polynucleotide phosphorylase (PNPase) inhibition, and the possible protection effects on mitochondrial DNA (mtDNA). Methods Human hepatocellular cell lines SK-Hep1 and Hep G2 and human bone sarcoma cell line U2OS were transfected by PNPase shRNA lentivirus with green fluorescent protein (GFP). PNPase expression was detected by Western blot. Then the mitochondria were separated, and MitomiRs were extracted. MitomiRs were analyzed by microRNA chips before and after PNPase shRNA treatment. MtDNA damage frequency was assessed by Q-PCR, and mitochondrial 8-hydroxydeoxyguanosine (8-OHdG) was detected by ELISA. Results Visible green fluorescence was found in SK-Hep1, HepG2 and U2OS cells transfected with PNPase shRNA lentivirus 12-24 hours later. When the MOI (transfection plural) was 20, the transfection efficiencies of 3 kinds of cells were more than 80%. In the next 2 consecutive weeks, the transfection efficiencies maintain