【摘要】 目的 探讨长链非编码RNA转移相关肺腺癌转录本1(lncRNA MALAT1)与微小RNA-874(miR-874)在血小板衍生因子(PDGF)-BB处理的气道平滑肌细胞(ASMCs)中的表达与功能。方法 采用PDGF-BB处理ASMCs建立哮喘细胞模型。将ASMCs分为正常培养的对照组(Control组)、PDGF-BB处理的PDGF-BB组[再根据处理时间分为PDGF-BB(6 h)组、PDGF-BB(12 h)组、PDGF-BB(28 h)组、PDGF-BB(24 h)组)]、PDGF-BB处理的转染sh-MALAT组(PDGF-BB+sh-MALAT组)、PDGF-BB处理的转染sh-NC组(PDGF-BB+sh-NC组)、PDGF-BB处理的转染miR-874 mimics组(PDGF-BB+miR-874 mimics组)、PDGF-BB处理的转染miR-NC组(PDGF-BB+miR-NC组)、PDGF-BB处理的共转染sh-NC与miR-NC组(PDGF-BB+sh-NC+miR-NC组)、PDGF-BB处理的共转染sh-MALAT1与miR-NC组(PDGF-BB+sh-MALAT1+miR-NC组)、PDGF-BB处理的共转染sh-MALAT1与miR-874 inhibitor组(PDGF-BB+sh-MALAT1+miR-874 inhibitor组)。将MALAT-WT或MALAT-MUT序列和miR-874 mimics或miR-NC共同转染入psi-CHECK2荧光素酶报告载体中，并将共转染的细胞分为miR-874 mimics+MALAT-WT组、miR-NC+MALAT-WT组、miR-874 mimics+MALAT-MUT组和miR-NC+MALAT-MUT组。采用RT-PCR检测MALAT1、miR-874表达水平，采用CCK-8法检测吸光度值代表细胞增殖能力，采用Transwell实验计算迁移细胞数代表细胞迁移能力，采用ELISA试验检测IL-1β、IL-6和肿瘤坏死因子(TNF)-α水平。使用在线数据库starbase预测MALAT和miR-874之间的潜在结合位点，采用双荧光素酶报告系统检测各组ASMCs中荧光素酶活性。结果 Control组、PDGF-BB(6 h)组、PDGF-BB(12 h)组、PDGF-BB(18 h)组及PDGF-BB(24 h)组细胞MALAT1相对水平依次升高，miR-874相对水平依次降低(P<0.05)。与Control组比较，PDGF-BB组、PDGF-BB+sh-NC组及PDGF-BB+sh-MALAT1组、PDGF-BB+miR-NC组及PDGF-BB+miR-874 mimics组细胞增殖能力、迁移能力及细胞上清液中IL-1β、IL-6和肿瘤坏死因子(TNF)-α水平均明显增强；与PDGF-BB组比较，PDGF-BB+sh-MALAT1组及PDGF-BB+miR-874 mimics组细胞上述能力与指标水平均明显降低(P<0.05)。与miR-NC+MALAT1-WT组细胞比较，miR-874 mimics+MALAT1-WT组细胞荧光素酶活性显著降低；与Control组比较，转染sh-MALAT1后细胞miR-874表达水平明显升高(P<0.05)。与sh-NC+miR-NC组比较，PDGF-BB+sh-NC+miR-NC组、PDGF-BB+sh-MALAT1+miR-NC组和PDGF-BB+sh-MALAT1+miR-874 inhibitor组细胞增殖能力与迁移能力均明显升高；与PDGF-BB+sh-NC+miR-NC组比较，PDGF-BB+sh-MALAT1+miR-874 inhibitor组细胞上述能力均明显降低(P<0.05)。结论 通过降低细胞增殖、迁移和炎症因子分泌可抑制MALAT1/miR-874轴表达，在PDGF-BB诱导的ASMCs中发挥保护作用。更多还原

【Abstract】 Objective To investigate the expression and function of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1(lncRNA MALAT1) and microRNA-874(miR-874) in platelet-derived factor(PDGF)-BB treated airway smooth muscle cells(ASMCs).Methods The asthma cell model was established by treating ASMCs with PDGF-BB.ASMCs were divided into normal culture control group and PDGF-BB treated group[which were divided into PDGF-BB(6 h) group, PDGF-BB(12 h) group, PDGF-BB(28 h)group and PDGF-BB(24 h) group according to the treatment time],transfected sh-MALAT group with PDGF-BB treatment(PDGF-BB+sh-MALAT group),sh-NC group with PDGF-BB treatment(PDGF-BB+sh-NC group),miR-874 mimics group with PDGF-BB treatment(PDGF-BB+miR-874 mimics group),PDGF-BB transfected miR-NC group(PDGF-BB+miR-NC group),the combined transfection of sh-NC and miR-NC group with PDGF-BB treatment(PDGF-BB+sh-NC+miR-NC group),the combined transfection of sh-MALAT1 and miR-NC group with PDGF-BB treatment(PDGF-BB+sh-MALAT1+miR-NC group) the combined tronsfection of sh-MALAT1 and miR-874 inhibitor group with PDGF-BB treatment(PDGF-BB+sh-MALAT1+miR-874 inhibitor group).MALAT-WT or MALAT-MUT sequence and miR-874 mimics or miR-NC were co-transfected into the psi-CHECK2 luciferase reporter vector.The co-transfected cells were divided into miR-874 mimics+MALAT-WT group, miR-NC+MALAT-WT group, miR-874 mimics+MALAT-MUT group and miR-NC+MALAT-MUT group.The expression levels of MALAT1 and miR-874 were detected by RT-PCR.The absorbance value was detected by CCK-8 method, which represented the cell proliferation ability.The number of migrating cells was calculated by transwell experiment to represent the migration ability of cells.The levels of IL-1β,IL-6 and TNF-α were detected by ELISA.The online database starbase was used to predict potential binding sites between MALAT and miR-874.Double luciferase reporting system was used to detect luciferase activity in ASMCs of each group.Results The relative level of MALAT1 in control group, PDGF-BB(6 h) group, PDGF-BB(12 h) group, PDGF-BB(18 h) group and PDGF-BB(24 h) group were increased successively, and the relative level of miR-874 were decreased successively(P<0.05).Compared with control group, the proliferation and migration of cells and the levels of IL-1β,IL-6 and TNF-α in cell supernatant in PDGF-BB group, PDGF-BB+sh-NC group and PDGF-BB+sh-MALAT1 group, PDGF-BB+miR-NC group and PDGF-BB+miR-874 mimics group were significantly enhanced; compared with PDGF-BB group, the above abilities and indexes levels of PDGF-BB+sh-MALAT1 group and PDGF-BB+miR-874 mimics group were significantly decreased(P<0.05).Compared with miR-NC+MALAT1-WT group, the luciferase activity in miR-874 mimics+MALAT1-WT group was significantly decreased; compared with the control group, the expression level of miR-874 in cells transfected with sh-MALAT1 was significantly increased(P<0.05).Compared with sh-NC+miR-NC group, cell proliferation and migration ability of PDGF-BB+sh-NC+miR-NC group, PDGF-BB+sh-MALAT1+miR-NC group and PDGF-BB+sh-MALAT1+miR-874 inhibitor group were significantly increased; compared with PDGF-BB+sh-NC+miR-NC group, the above ability of PDGF-BB+sh-MALAT1+miR-874 inhibitor group were significantly decreased(P<0.05).Conclusion Inhibition of MALAT1/miR-874 axis expression can play a protective role in PDGF-BB-induced ASMCs by reducing cell proliferation, migration and secretion of inflammatory factors.更多还原