摘要
目的探讨miRNA-20a在脂多糖(lipopolysaccharide,LPS)诱导的A549细胞焦亡与炎症反应中的作用与调控机制。方法培养人肺泡上皮A549细胞,以LPS为刺激物建立细胞焦亡及炎症反应模型,给予LPS+三磷酸腺苷刺激为LPS组,未给予刺激为空白对照组,验证模型是否成功。将A549细胞根据转染物质分为miRNA-20a模拟物(mimics)组、mimics-阴性对照(negative control,NC)组、miRNA-20a抑制物(inhibitor)组和inhibitor-NC组,构建过表达及沉默miRNA-20a的A549细胞模型,各组细胞在转染24 h后给予LPS+三磷酸腺苷刺激。构建TLR4-3'UTR双荧光素酶报告基因载体质粒,包括野生型(WT)载体质粒TLR4-3'UTR-WT1、TLR4-3'UTR-WT2及相应的变异型(MUT)载体质粒TLR4-3'UTR-MUT1、TLR4-3'UTR-MUT2,用双荧光素酶报告基因实验验证miRNA-20a的靶基因。采用实时荧光定量聚合酶链反应、免疫印迹试验及酶联免疫吸附试验检测各组焦亡标志因子消皮素D(gsdermin D,GSDMD),炎症因子凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶1(cysteinyl aspartate specific proteinase-1,Caspase-1)、白细胞介素1β(interleukin-1β,IL-1β),以及信号分子Toll样受体4(Toll-like receptor-4,TLR4)、核因子κB(nuclear factor-κB,NF-κB)mRNA及蛋白表达。免疫荧光检测各组细胞TLR4表达和NF-κB入核情况。结果LPS组A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1βmRNA和蛋白表达量均高于空白对照组(P<0.05),模型建立成功。mimics组miRNA-20a表达量高于mimic-NC组(P<0.05),inhibitor组miRNA-20a表达量低于inhibitor-NC组(P<0.01)。双荧光素酶报告基因实验显示,TLR4-3'UTR-WT与miRNA-20a mimics共转染组的相对荧光值低于TLR4-3'UTR-WT与mimics-NC共转染组(P<0.05)。mimics组LPS介导的A549细胞GSDMD、ASC、NLRP3、Caspase-1、IL-1β、TLR4、NF-κB
Methods Cultured human alveolar epithelial A549 cells were assigned into LPS group and blank control group.LPS group was stimulated with LPS and adenosine triphosphate to induce pyroptosis and inflammation.A549 cells were divided into 4 groups:miR-20a mimics group,mimics-negative control(NC)group,inhibitor group and inhibitor-NC group.MiRNA-20a mimics,mimics-NC,inhibitor,and inhibitor-NC were transfected respectively into A549 cells,and after 24 h,the cells were collected to verify transfection efficiency by qPCR.MiRNA-20a mimics and the constructed TLR4-3'UTR double luciferase reporter plasmid were co-transfected into A549 cells,and luciferase activity was analyzed.MiRNA-20a mimics/inhibitors were transfected into A549 cells,and then the cells were stimulated by LPS for 8 h followed by adenosine triphosphate for 30 min.QPCR,Western Blot and ELISA were used to detect the expression of GSDMD,inflammatory factors(ASC,NLRP3,Caspase-1,IL-1β)and Signaling molecules(TLR4、NF-κB)in A549 cells at mRNA level and protein level.Immunofluorescence was used to detect the expression of TLR4 in the A549 cells and NF-κB in the nucleus of A549 cells after transfecting with miRNA-20a mimics/inhibitor.Results The mRNA and protein expression of pyroptosis marker molecule(GSDMD)and inflammatory factors(ASC,NLRP3,Caspase-1,IL-1β)in A549 cells stimulated with LPS were significantly higher than those in the blank control group,and the differences were statistically significant(P<0.05).The expression of miRNA-20 in the mimics group was significantly higher than that in the mimic-NC group(P<0.05),while the expression of miRNA-20a in the inhibitor group was lower than that in the inhibitor-NC group(P<0.01).The double luciferase reporter gene experiment showed that the relative fluorescence value of the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics was significantly lower than the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics-NC(P<0.05).The mRNA and protein levels of pyroptosis marker molecule(GSDMD),infla
作者
陶慧娴
王木子
郭艳
邹芸苏
卢志涛
丁溢芳
周晓光
许卫东
Tao Huixian;Wang Muzi;Guo Yan;Zou Yunsu;Lu Zhitao;Ding Yifang;Zhou Xiaoguang;Xu Weidong(Department of Pediatrics,Zhangjiagang First People's Hospital,Suzhou 215600,China;Neonatal Medical Center,Children's Hospital of Nanjing Medical University,Nanjing 210008,China)
出处
《中华新生儿科杂志（中英文）》
CAS
CSCD
2023年第2期107-114,共8页
Chinese Journal of Neonatology
基金
苏州市产业技术创新专项(SYSD2017005)
张家港市卫计系统青年科技项目(ZJGQNKJ201703)。
