下调微小RNA-19a对胶质瘤细胞侵袭能力的影响及机制

Effect of microRNA-19a down-regulation on invasiveness of glioma cells and the potential mechanism

目的 探讨下调微小RNA（miR）-19a的表达对人脑胶质瘤细胞株U87侵袭能力的影响及其机制.方法 采用实时荧光定量聚合酶链反应（FQ-PCR）检测转染miR-19a抑制物的效率.转染后48 h,采用Western blot法检测U87细胞中多亮氨酸重复区免疫球蛋白样蛋白1（LRIG1）的表达,并通过Transwell实验检测U87细胞的侵袭能力.构建报告质粒,于转染后72 h用荧光素酶报告实验验证miR-19a和LRIG1的相互作用.结果 FQ-PCR结果显示转染miR-19a抑制物后,U87中miR-19a的表达量较对照组降低了（78.2±5.1）％,差异有统计学意义（P＜0.01）.转染miR-19a抑制物后,U87穿膜细胞数较对照组明显减少,分别为（25.9±3.9）个和（85.3±6.1）个（P＜0.01）.荧光素酶报告实验结果显示,下调miR-19a后野生型报告质粒的荧光素酶活性较对照组升高（4.89±0.26）倍（P＜0.01）,而突变型质粒的荧光素酶活性较对照组无明显变化.Transwell实验结果表明在下调miR-19a后,LRIG1沉默组的穿膜细胞数较对照组明显增加,分别为（47.8±3.1）个和（22.7±4.2）个（P＜0.05）.结论 下调miR-19a可以抑制U87细胞的侵袭力,其机制可能是上调LRIG1的表达.

Objective To explore whether down-regulation of microRNA （miR）-19a could inhibit invasion of glioma cells and the potential mechanism.Methods The transfection efficiency was determined by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）.The expression of leucine-rich repeats and immunoglobulin-like domains 1 （LRIG1） was detected by Western blotting and the invasion ability of U87 cells was evaluated by Transwell assay at 48 h post-transfection.Luciferase reporter assay was performed to identify the interaction between miR-19a and LRIG1 at 72 h post-transfection.Results FQ-PCR demonstrated that the expression of miR-19a was drastically decreased by （78.2 ±5.1）% following miR-19a inhibition （P〈 0.01）.The number of invasive cells in antago miR-19a group was obviously less than that in control group （25.9 ± 3.9 vs.85.3 ± 6.1,P 〈 0.01）.Following down-regulation of miR-19a,the luciferase activity of wild reporter was increased by （4.89 ± 0.26） fold （P 〈 0.01） compared to that of control,whereas the luciferase activity of mutant reporter was almost unaffected.The number of invasive cells in experimental group （antagomiR-19a ± siLRIG1） was significantly greater than in control group （antagomiR-19a ± scramble） （47.8 ± 3.1 vs.22.7 ± 4.2,P 〈 0.05）.Conclusion Down-regulation of miR-19a could suppress invasion of U87cells,which may be mediated by LRIG1.