长链非编码RNA TUG1对冷保存小鼠肝脏细胞的影响及其机制

Long Noncoding RNA TUG1 Protects Against Cold Storage Injury of Mouse Liver Cells by Blocking Mitochondrial Apoptosis and Endoplasmic Reticulum Stress Pathways

探讨长链非编码RNA TUG1对冷保存小鼠肝脏细胞的影响及其作用机制。方法:构建小鼠肝细胞(HC)及肝窦内皮细胞(LSEC)冷保存损伤模型,利用qPCR检测TUG1的表达;将肝脏细胞分为两组TUG1慢病毒组(lenti-TUG1)和慢病毒阴性对照组(lenti-control),TUG1慢病毒组通过慢病毒感染方式上调TUG1的表达,构建细胞冷保存损伤模型,分别采用ELISA、CCK-8法及流式技术检测培养液中LDH水平、细胞活性及细胞凋亡率,Western blot法检测线粒体凋亡通路相关蛋白Bax、Bcl-2、Cyt-C、cleaved caspase-9、cleaved caspase-3 和cleaved PARP以及内质网应激通路相关蛋白GRP78、PERK、p-eIF2α、CHOP、cleaved-caspase 12和cleaved-PARP的表达。结果:TUG1在冷保存的肝脏细胞中表达下调;过表达TUG1可降低LDH水平及凋亡率,增加细胞活性,降低线粒体凋亡相关蛋白及内质网应激相关蛋白表达。结论:TUG1可能通过线粒体凋亡和内质网应激通路抑制细胞凋亡来实现对冷保存诱导细胞损伤的保护作用。

To investigate the protective effect of the long noncoding RNA TUG1 on cold storage injury of mouse hepatocytes and liver sinusoidal endothelial cells and its possible mechanism. Methods A cold preservation model of mouse hepatocytes(HC) and liver sinusoidal endothelial cell(LSEC) was established and the expression of TUG1 was detected by qPCR.The expression of TUG1 was up-regulated by lentivirus infection, and the cold preservation injury model was prepared. The concentration of lactate dehydrogenase(LDH) in the culture media was detected by ELISA. Cells vitality was investigated by CCK-8 assay. Apoptosis was detected by flow cytometry analysis. The expression of mitochondrial apoptosis associated protein Bax, Bcl-2, cytochrome C, cleaved caspase-9, cleaved caspase-3 and cleaved PARP was observed by Western blot analysis.The expression of endoplasmic reticulum stress associated protein GRP78, PERK, p-eIF2α, CHOP, cleaved-caspase 12 and cleaved-PARP was also observed by Western blot analysis. Results The expression of TUG1 is down-regulated in cold preserved liver cells.Over expression of TUG1 can reverse the increased LDH level and apoptosis rate induced by cold storage injur and the increased expression of mitochondrial apoptosis related proteins and endoplasmic reticulum stress related proteins. Conclusion The long noncoding RNA TUG1 protects against cold storage injury of mouse livers by blocking mitochondrial apoptosis and endoplasmic reticulum stress pathways.