CAJ | 학술논문

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.
목적 탐토하조미소RNA(miR)-19a적표체대인뇌효질류세포주U87침습능력적영향급기궤제.방법 채용실시형광정량취합매련반응(FQ-PCR)검측전염miR-19a억제물적효솔.전염후48 h,채용Western blot법검측U87세포중다량안산중복구면역구단백양단백1(LRIG1)적표체,병통과Transwell실험검측U87세포적침습능력.구건보고질립,우전염후72 h용형광소매보고실험험증miR-19a화LRIG1적상호작용.결과 FQ-PCR결과현시전염miR-19a억제물후,U87중miR-19a적표체량교대조조강저료(78.2±5.1)％,차이유통계학의의(P＜0.01).전염miR-19a억제물후,U87천막세포수교대조조명현감소,분별위(25.9±3.9)개화(85.3±6.1)개(P＜0.01).형광소매보고실험결과현시,하조miR-19a후야생형보고질립적형광소매활성교대조조승고(4.89±0.26)배(P＜0.01),이돌변형질립적형광소매활성교대조조무명현변화.Transwell실험결과표명재하조miR-19a후,LRIG1침묵조적천막세포수교대조조명현증가,분별위(47.8±3.1)개화(22.7±4.2)개(P＜0.05).결론 하조miR-19a가이억제U87세포적침습력,기궤제가능시상조LRIG1적표체.
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.