摘要
目的筛选并确定调控miR-592作用的关键lncRNA,探讨其对miR-592沉默靶基因cep135的调控作用。方法利用lncRNA基因芯片筛选小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)及KO miR-592 mESCs中差异的lncRNA从而找出关键调控分子lncRNA-135。利用转染试剂干扰lncRNA-135的表达,使用Western印迹法、双荧光素酶报告系统及免疫细胞化学染色等技术检测lncRNA-135对miR-592及其靶基因cep135的表达影响,进而阐明lncRNA-135对mESCs多能性的调控作用。结果lncRNA基因芯片分离确定了527条差异表达的lncRNA,经过生物信息学分析及qRT-PCR验证,确定了评分最高的lncRNA-135为关键作用分子。双荧光素酶报告系统证实了lncRNA-135能够结合miR-592以干扰其沉默靶基因cep135。而Western印迹法和免疫细胞化学染色结果则证实lncRNA-135通过作用于miR-592,阻止其对cep135的抑制作用,进而促进mESCs的多能性。结论lncRNA-135可以通过充当miR-592的“分子海绵”从而调节与mESCs多能性维持有关的多个基因的表达,这将可能帮助建立调控性miRNA网络与mESCs多能性之间的联系。
Objective To screen and identify key lncRNAs that regulate the function of miR-592 and its mechanisms.Methods The key regulatory molecules that regulate miR-595 were screened and identified with lncRNA chip in mouse embryonic stem cells(mESCs)of wild-type and miR-592 knockout(KO)mice.The expression of lncRNA-135 was knocked down by transfection of interference fragments.The effect of lncRNA-135 on the expression of miR-592 and its target gene cep135 was confirmed by Western blot,dual luciferase reporter system and immunocytochemistry.Furthermore,the regulation of mESCs pluripotency by lncRNA-135 was verified.Results Through lncRNA gene chip screening,527 differentially expressed lncRNAs were identified.After bioinformatics analysis and qRT-PCR verification,the lncRNA-135 with the highest score was identified as the key lncRNA.The dual luciferase reporter system confirmed that lncRNA-135 was able to bind miR-592 to interfere with its silencing target gene cep135.Western blot and immunocytochemical results confirmed that lncRNA-135 promoted the pluripotency of mESCs by inhibiting the miR-592-cep135 system.Conclusion The results suggest that lncRNA-135 can act as a sponge for miR-592 to regulate the expression of multiple genes related to the maintenance of mESCs pluripotency.
作者
李培昕
张兴燎
周杨
张宁彦
张敬
张军
LI Pei-xin;ZHANG Xing-liao;ZHOU Yang;ZHANG Ning-yan;ZHANG Jing;ZHANG Jun(Tongji University School of Medicine,Shanghai 200092,China;Tongji University School of Life Science and Technology,Shanghai 200092,China)
出处
《同济大学学报（医学版）》
CAS
2020年第5期552-560,共9页
Journal of Tongji University(Medical Science)
基金
国家自然科学基金面上项目(81771417)。
