miR-146a对食管鳞癌细胞生物学行为的影响及其机制

Effect of miR-146a on biological behavior of esophageal squamous cell carcinoma cells and its mechanism

目的探讨miR-146a对食管鳞癌细胞增殖、侵袭、迁移、凋亡及细胞周期等生物学行为的影响及其机制。方法应用实时荧光定量聚合酶链反应(RT-qPCR)技术检测miR-146a在食管鳞癌细胞株Eca109、KYSE140和KYSE150中的表达。将miR-146a模拟物转染Eca109细胞,上调miR-146a的表达,采用细胞增殖实验检测细胞的增殖能力,Transwell实验检测细胞的侵袭能力,划痕实验检测细胞的迁移能力,流式细胞术检测细胞的凋亡和细胞周期。运用相关生物信息学技术预测miR-146a的靶基因,采用双荧光素酶报告基因实验检测miR-146a与靶基因白介素1受体相关激酶1(IRAK1)3'端非翻译区(3TTR)的结合情况,RT-qPCR技术和Western blot法分别检测靶基因mRNA和蛋白的表达。结果食管鳞癌Ecal09、KYSE140和KYSE150细胞中miR-146a的相对表达量分别为0.36±0.05、0.16±0.06和0.09±0.02,均低于食管正常上皮细胞HEEC(1.00±0.05,均P 0.01)。miR-146a模拟物转染Ecal09细胞后,细胞的吸光度值、穿膜细胞数[(52±18)个]、划痕减少距离[48h为(25.29±0.77)μm,72h为(30.66±0.91)μm]均明显低于阴性对照组和空白对照组(均P 0.01),早期凋亡率[(6.13±0.91)%]高于阴性对照组[(2.50±0.68)%,P 0.01]和空白对照组[(1.70±0.20)%,P 0.01];G,期细胞百分比[(44.74±6.76)%]较阴性对照组和空白对照组减少(均P 0.05),G2/M期细胞百分比[(41.88±2.88)%]较阴性对照组和空白对照组增多(均P 0.01)。双荧光素酶报告基因实验结果显示,共转染IRAKIWT和miR-146a模拟物组的荧光素酶活性明显低于其他对照组(均P 0.01)。RT-qPCR和Western blot检测结果显示,miK-146a模拟物转染组细胞中IRAKImRNA的表达水平为1.02±0.28,蛋白的表达水平为1.00±0.05,均低于阴性对照组和空白对照组(均/0.01)。结论miR-146a在食管鳞癌细胞株中表达降低,扮演着抑癌基因的角色。miR-146a能抑制食管鳞癌细胞的增殖、侵袭和迁移,促进其凋亡,阻滞细胞周期于G2/M期。miR-146a可能通过IRAKI的介导调控食管癌细胞的恶性生物学行为。

Objective Fo investigate the effects and mechanisms of miR-146a on the proliferation,invasion,migration,apoptosis and cell cycle of esophageal squamous cell carcinoma cells.Methods The expressions of miR-146a in 3 esophageal squamous carcinoma cell lines(Eca109,KYSE140,KYSE150)were detected by real-time quantitative polymerase chain reaction(RT-q PCR).MiR-146a mimics was transfected into Ecal09 to up-regulate the expression of miR-146a.Effects of miR-146a on cell proliferation,invasion and migration were evaluated by cell counting kit 8(CCK-8),Transwell assay and wound-healing assay,respectively.The cell apoptosis and cycle were assessed by flow cytometry(FCM).Finally,relevant bioinformatics techniques were used to predict the target gene of miR-146a.Dual luciferase reporter assay was used to identify the interaction of 3'terminal untranslated region(3 r UTR)of miR-146a and its target gene,interleukin-1 receptor-associated kinase(IRAKI).RT-qPCR and western blot were used to detect the mRNA and protein expressions of IRAKI,respectively.Results The relative expressions of miR-146a in 3 esophageal squamous cell carcinoma cells(Eca 109,KYSE140,KYSE150)were 0.36±0.05,0.16±0.06 and 0.09±0.02,respectively,all of which were significantly lower than 1±0.05 of normal esophageal epithelial cells(HEEC)(P<0.01).The model of esophageal squamous cell carcinoma cells was constructed by transfecting miH-146a mimics into Ecal09 cells.The results showed that the ability of absorbance value,the number of transniemhrane cells(52±18),the reduced scratch distances at 48 hours and 72 hours after transfection[(25.29±0.77)μm,(30.66±0.91)μm]were significantly lower than those of the negative control group and blank control group(all P<0.01).The early apoptosis rate was(6.13±0.91)%,higher than(2.50±0.68)%of the negative control group(P<0.01)and(1.70±0.20)%of blank control group(P<0.01).The percentage of cells in G,phase[(44.74±6.76)%]was decreased while the G2/M phase[(41.88±2.88)%]was increased when compared with the negative control group and the blank control group(P<0.05 and P<0.01,respectively).The results of dual luciferase reporter gene assay showed that luciferase activity in the group co-transfected with IRAKI-wild-type and miR-146a mimics was significantly lower than that in the control groups(P<0.01).The results of RT-qPCH and western blot showed that the mRNA and protein expressions of IRAKI in the co-transfected group were 1.02±0.28 and 1.00±0.05,respectively,both lower than those in the negative control group and the blank control group(P<0.01).Conclusions The expressions of miR-146a are decreased in the esophageal squamous cell lines,which plays a role as tumor suppressor gene.MiR-146a can inhibit the proliferation,invasion and migration of esophageal squamous cell cells,promote apoptosis,and block the cell cycle at G2/M stage.MiR-146a may mediate the malignant biological behavior of esophageal cancer cells through the regulation of IRAKI.