摘要目的 以烟雾吸人性损伤为前提,探讨在香烟烟雾提取物(CSE)刺激下转染微小RNA-146a对人肺腺癌A549细胞中Fas相关因子2(FAF-2)及炎症因子的影响. 方法 (1)构建pMIR-FAF-2重组质粒、pMIR-FAF-2重组突变质粒.将第3代人胚胎肾293(HEK-293)细胞按随机数字表法分为3组,每组5孔:质粒+微小RNA对照组,转染pMIR-FAF-2重组质粒、pRL-TK质粒及微小RNA对照剂;质粒+微小RNA-146a组,转染pMIR-FAF-2重组质粒、pRL-TK质粒及微小RNA-146a模拟物;突变质粒+微小RNA-146a组,转染pMIR-FAF-2重组突变质粒、pRL-TK质粒及微小RNA-146a模拟物.培养24 h后,采用双荧光素酶报告基因检测细胞相对荧光素酶活性.(2)将第3代A549细胞按随机数字表法分为3组,每组4孔:微小RNA对照组,转染微小RNA对照剂;微小RNA-146a增强组,转染微小RNA-146a模拟物;微小RNA-146a抑制组,转染微小RNA-146a抑制剂.培养24 h后,采用实时荧光定量RT-PCR法检测细胞微小RNA-146a表达及FAF-2的mRNA表达.(3)将第3代A549细胞同实验(2)分组及处理24 h后,采用体积分数0.8％的CSE刺激24 h.采用实时荧光定量RT-PCR法检测细胞FAF-2、IL-8、单核细胞趋化蛋白1(MCP-1)、生长调节致癌基因α(GRO-α)的mRNA表达量,ELISA法检测细胞培养上清液中IL-8、MCP-1、GRO-α的蛋白表达量,采用蛋白质印迹法检测环氧化酶2(COX-2)的蛋白表达量.对数据行单因素方差分析、LSD检验.结果 (1) pMIR-FAF-2重组质粒、pMIR-FAF-2重组突变质粒确认构建成功.质粒+微小RNA对照组与突变质粒+微小RNA-146a组HEK-293细胞相对荧光素酶活性相近(P＞0.05),质粒+微小RNA-146a组HEK-293细胞相对荧光素酶活性明显低于质粒+微小RNA对照组和突变质粒+微小RNA-146a组(P值均小于0.01).(2)微小RNA对照组与微小RNA-146a抑制组A549细胞微小RNA-146a表达量相近(P＞0.05),均明显低于微小RNA-146a增强组(P值均小于0.01).微小RNA对照组和微小RNA-146a抑制组A549细胞FAF-2的mRNA表达量相近(P＞0.05),均明显高于微小RNA-146a增强组(P值均小于0.05).(3) CSE刺激后,微小RNA对照组与微小RNA-146a抑制组A549细胞FAF-2的mRNA表达量分别为1.46±0.21和1.43±0.34,二者相近(P＞0.05),均明显高于微小RNA-146a增强组的0.57±0.11(P值均小于0.05).微小RNA-146a增强组A549细胞IL-8、MCP-1、GRO-α的mRNA表达量均明显低于微小RNA对照组和微小RNA-146a抑制组(P值均小于0.01),微小RNA-146a抑制组A549细胞IL-8、MCP-1、GRO-α的mRNA表达量明显高于微小RNA对照组(P值均小于0.05);微小RNA-146a增强组A549细胞培养上清液中IL-8、MCP-1、GRO-α的蛋白表达量均明显低于微小RNA对照组和微小RNA-146a抑制组(P值均小于0.05);微小RNA-146a抑制组A549细胞培养上清液中IL-8蛋白表达量与微小RNA对照组相近(P＞0.05),MCP-1、GRO-α蛋白表达量明显低于微小RNA对照组(P值均小于0.05).微小RNA-146a增强组A549细胞COX-2蛋白表达量明显低于微小RNA对照组和微小RNA-146a抑制组(P值均小于0.05),微小RNA对照组A549细胞COX-2蛋白表达量与微小RNA-146a抑制组相近(P＞0.05).结论 CSE刺激下转染微小RNA-146a的A549细胞中,微小RNA-146a可通过与靶基因FAF-2的3'端非翻译区结合,降低FAF-2的表达,从而降低炎症因子表达量.

abstractsObjective Under the premise of smoke inhalation injury,to explore the effects of microRNA-146a on Fas-associated factor 2 (FAF-2) and inflammatory factors in human lung adenocarcinoma A549 cells under the stimulation of cigarette smoke extract (CSE).Methods (1) The pMIR-FAF-2 recombinant plasmid and the pMIR-FAF-2 recombinant mutated plasmid were constructed.Human embryonic kidney 293 (HEK-293) cells of the third passage were divided into 3 groups according to the random number table,with 5 wells in each group.Cells in plasmid + microRNA control group were transfected with pMIR-FAF-2 recombinant plasmid,pRL-TK plasmid,and microRNA control;cells in plasmid + microRNA-146a group were transfected with pMIR-FAF-2 recombinant plasmid,pRL-TK plasmid,and microRNA-146a mimics;cells in mutated plasmid + microRNA-146a group were transfected with pMIR-FAF-2 recombinant mutated plasmid,pRL-TK plasmid,and microRNA-146a inhibitor.After culture for 24 h,the relative luciferase activity in cells was assessed by dual-luciferase reporter gene assay.(2) Human lung adenocarcinoma A549 cells of the third passage were divided into 3 groups according to the random number table,with 4 wells in each group.Cells in microRNA control group were transfected with microRNA control;cells in microRNA-146a enhancement group were transfected with microRNA-146a mimics;cells in microRNA-146a inhibition group were transfected with microRNA-146a inhibitor.After culture for 24 h,the mRNA expression levels of microRNA-146a and FAF-2 in cells were determined with real-time fluorescent quantitative reverse transcription-PCR.(3) A549 cells of the third passage were stimulated by 0.8％ CSE for 24 h after being divided and treated with the same method used in experiment (2).The mRNA expression levels of FAF-2,IL-8,monocyte chemotactic protein-1 (MCP-1),and growth-regulated oncogene-α (GRO-α) in cells were determined with real-time fluorescent quantitative reverse transcription-PCR.The protein expression levels of IL-8,MCP-1,and GRO-α in A549 cell culture supernatant were determined by enzyme-linked immunosorbent assay.The protein expression level of cyclooxygenase 2 (COX-2) of cells was assessed by Western blotting.Data were processed with one-way analysis of variance and LSD test.Results (1) The pMIR-FAF-2 recombinant plasmid and pMIR-FAF-2 recombinant mutated plasmid were confirmed with successful construction.The relative luciferase activity in HEK-23 cells of plasmid + microRNA control group was close to that of mutated plasmid + microRNA-146a group (P ＞ 0.05).The relative luciferase activity in HEK-23 cells of plasmid + microRNA-146a group was significantly lower than that of plasmid + microRNA control group and mutated plasmid + microRNA-146a group (with P values below 0.01).(2) The expression level of microRNA-146a in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P ＞ 0.05),and they were both significantly lower than the expression level of microRNA-146a in A549 cells of microRNA-146a enhancement group (with P values below 0.01).The mRNA expression level of FAF-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P ＞ 0.05),and they were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (with P values below 0.05).(3) After stimulation of CSE,the mRNA expression level of FAF-2 in A549 cells of microRNA control group (1.46 ± 0.21) was close to that of microRNA-146a inhibition group (1.43 ± 0.34,P ＞ 0.05),which were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (0.57 ± 0.11,with P values below 0.05).The mRNA expression levels of IL-8,MCP-1,and GRO-α in A549 cells of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.01).The mRNA expression levels of IL-8,MCP-1,and GRO-α in A549 cells of microRNA-146a inhibition group were significantly higher than those of microRNA control group (with P values below 0.05).The protein expression levels of IL-8,MCP-1,and GRO-α in A549 cell culture supernatant of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.05).The protein expression level of IL-8 in A549 cell culture supernatant of microRNA-146a inhibition group was close to that of microRNA control group (P ＞ 0.05),while the protein expression levels of MCP-1 and GRO-α in A549 cell culture supernatant of microRNA-146a inhibition group were significantly lower than those of microRNA control group (with P values below 0.05).The protein expression level of COX-2 in A549 cells of microRNA-146a enhancement group was significantly lower than the levels of microRNA control group and microRNA-146a inhibition group (with P values below 0.05).The protein expression level of COX-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P ＞ 0.05).Conclusions In A549 cells,after being transfected with microRNA-146a and stimulated by CSE,microRNA-146a can decrease the expression of FAF-2 through integrating with the 3'-untranslated region of target gene FAF-2,thereby decrease the expression of inflammatory factors.