摘要
目的探讨微小RNA-365(miR-365)对脓毒症血管内皮细胞增殖、凋亡的影响及其作用机制。方法体外培养人脐静脉内皮细胞(HUVECs),利用脂多糖(LPS)诱导HUVECs模拟炎性环境,采用实时荧光定量聚合酶链反应(qRT-PCR)与蛋白免疫印迹法(Western blot)检测miR-365、白细胞介素-1受体相关激酶1(IRAK1)的表达。实验分组:空白组、LPS组、LPS+miR-con组、LPS+miR-365组、LPS+miR-365+pcDNA组、LPS+miR-365+pcDNA-IRAK1组。甲基噻唑基四唑(MTT)检测6组细胞存活率;流式细胞术检测6组细胞凋亡率。采用酶联免疫吸附法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)含量。双荧光素酶报告实验验证miR-365与IRAK1的靶向调控关系;Western blot检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)蛋白表达。结果与空白组比较,LPS组HUVECs中miR-365的表达水平显著降低(P<0.05),IRAK1 mRNA及蛋白表达水平显著升高(P<0.05),HUVECs存活率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),Cleaved Caspase-3、Cleaved Caspase-9蛋白表达量显著升高(P<0.05),TNF-α、IL-1β、IL-6水平显著升高(P<0.05);与LPS+miR-con组比较,LPS+miR-365组HUVECs存活率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),Cleaved Caspase-3、Cleaved Caspase-9蛋白表达量均显著降低(P<0.05),TNF-α、IL-1β、IL-6水平显著降低(P<0.05);双荧光素酶报告实验证明miR-365可靶向调控IRAK1的表达;IRAK1过表达可部分逆转miR-365对LPS诱导的HUVECs增殖、凋亡及TNF-α、IL-1β、IL-6水平的影响。结论miR-365可通过靶向调控IRAK1的表达而影响血管内皮细胞增殖、凋亡及抑制炎性因子的分泌。
Objective To investigate the Effects of microRNA-365(miR-365)on proliferation and apoptosis of vascular endothelial cells in sepsis in vitro,and to explore its action mechanism.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro,and HUVECs were induced to induce inflammatory environment by lipopolysaccharide(LPS).Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expression levels of miR-365,interleukin-1 receptor-associated kinase 1(IRAK1).The experiment was divided into blank group,LPS group,LPS+miR-con group,LPS+miR-365 group,LPS+miR-365+pcDNA group,LPS+miR-365+pcDNA-IRAK1 group.Methylthiazolyl tetrazolium(MTT)was used to detect the survival rate of cells,and flow cytometry was used to detect the apoptosis rate.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).The dual luciferase reporter assay validated the targeted regulatory relationship between miR-365 and IRAK1.The expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 protein were detected by Western Blot.Results As compared with those in blank group,the expression levels of miR-365 in HUVECs of LPS group were significantly decreased(P<0.05),however,the expression levels of IRAK1 mRNA and protein were significantly increased(P<0.05),and the survival rate of HUVECs was significantly decreased(P<0.05).The apoptotic rate was significantly increased(P<0.05),the expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 protein were significantly increased(P<0.05),and the levels of TNF-α,IL-1βand IL-6 were significantly increased(P<0.05).As compared with that in LPS+miR-con group,the survival rate of HUVECs in LPS+miR-365 group was significantly increased(P<0.05),but,the apoptosis rate was significantly decreased(P<0.05),and the expression levels of Cleaved Caspase-3 and Cleaved Caspase-9 protein were significantly decreased(P<0.05).Moreover the levels of TNF-α,IL-1β,IL-6 were
作者
钟勇
贺珊珊
陈玉梅
ZHONG Yong;HE Shanshan;CHEN Yumei(Department of Critical Care Medicine,Yongchuan District TCM Hospital,Chongqing 402160,China)
出处
《河北医药》
CAS
2021年第2期171-175,181,共6页
Hebei Medical Journal
