摘要
目的探究环状RNA circLRP6、miR-5590-5p和C5aR1在肝衰竭中的保护作用及调控机制。方法通过D-半乳糖胺和脂多糖(D-galactosamine and LPS,D-GalN/LPS)诱导来构建体外肝衰竭细胞模型,流式细胞术检测L02细胞凋亡率,实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测circLRP6和miR-5590-5p的表达,Western blot法检测C5aR1蛋白的表达,RNA免疫共沉淀和双荧光素酶报告基因实验检测circLRP6和miR-5590-5p之间的结合作用,同时利用双荧光素酶报告基因实验检测C5aR1和miR-5590-5p的结合作用。结果与对照组比较,D-GalN/LPS诱导L02细胞凋亡率上升(P<0.05),circLRP6和C5aR1表达上调,miR-5590-5p表达下调(P<0.05)。同时,与D-GalN/LPS+miRNA-NC组比较,D-GalN/LPS+miR-5590-5p模拟物组的L02细胞凋亡率显著下降(P<0.05)。另外,双荧光素酶报告基因实验显示,circLRP6能够直接与miR-5590-5p结合,并且miR-5590-5p直接调控C5aR1。检测结果还发现,与D-GalN/LPS+shRNA组比较,D-GalN/LPS+sh-circLRP6组circLRP6基因和C5aR1蛋白表达下调。然而,与D-GalN/LPS+shRNA组比较,D-GalN/LPS+sh-circLRP6组L02细胞凋亡率显著下降。结论环状RNA circLRP6介导的miR-5590-5p具有改善肝衰竭的作用,其作用机制可能是通过靶向C5aR1来实现的。
Objective To investigate the protective effect and regulatory mechanism of circLRP6,miR-5590-5p and C5aR1 in liver failure.Methods The liver failure cell model in vitro was induced by D-galactosamine and LPS.The apoptosis rate of L02 cells was detected by flow cytometry(RT-qPCR).The expressions of circLRP6 and miR-5590-5p were detected by real-time quantitative PCR.The expression of C5aR1 protein was detected by Western blot.RNA immunoprecipitation and dual luciferase reporter gene assay were used to detect the binding between circLRP6 and miR-5590-5p.Dual luciferase report gene was applied to detect the binding between C5aR1 and miR-5590-5p.Results Compared with the control group,the apoptosis rate of L02 cells in the liver failure cell model increased(P<0.05).Compared with the control group,the expressions of circLRP6 and C5aR1 in the D-GalN/LPS group were up-regulated and the expression of miR-5590-5p was down-regulated(P<0.05).Meanwhile,compared with the D-GalN/LPS+miRNA-NC group,the apoptosis rate of L02 cells in the D-GalN/LPS+miR-5590-5p mimic group reduced significantly(P<0.05).In addition,dual luciferase reporter assay showed that circLRP6 could directly bind to miR-5590-5p,and miR-5590-5p directly regulated C5aR1.The tests also found that the expression of circLRP6 and C5aR1 were down-regulated in D-GalN/LPS+sh-circLRP6 group compared with D-GalN/LPS+shRNA group.Compared with D-GalN/LPS+shRNA group,the apoptosis rate of L02 cells in D-GalN/LPS+sh-circLRP6 group decreased significantly.Conclusion CircLRP6-mediated miR-5590-5p can improve liver failure,and its mechanism may be achieved by targeting C5aR1.
作者
陈玲
李楠
杨建乐
CHEN Ling;LI Nan;YANG Jianle(Department of Infectious Diseases,Zhejiang Hospital,Zhejiang 310013,China)
出处
《医学研究杂志》
2023年第6期54-59,共6页
Journal of Medical Research
基金
浙江省医药卫生科技计划项目(2021KY422,2022KY002)。
