CAJ | 학술논문

目的探讨miR-200b靶向调控PROM1的表达对胶质瘤细胞侵袭能力的影响以及miR-200b抑瘤的分子机制.方法构建PROM1 3’端非翻译区(3'UTR)荧光素酶报告载体,荧光素酶报告检测miR-200b对PROM1 3'UTR-荧光素酶活性的影响.将miR-200b模拟物转染胶质瘤U87细胞,采用实时荧光定量PCR和Western blot检测PROM1 mRNA和蛋白的表达水平.将PROM1 siRNA转染U87细胞,Transwell侵袭实验检测PROM1下调对U87细胞侵袭能力的影响.结果双荧光素酶报告检测显示,miR-200b能特异性地与PROM1 3'UTR结合,抑制其荧光素酶活性,其荧光素酶活性强度下降了57.0％,差异有统计学意义(P＜0.01).过表达miR-200b的U87细胞中PROM1蛋白和mRNA表达水平均降低,转染miR-200b组和对照组中PROM1 mRNA的表达水平分别为0.64 ±0.05和0.95 ±0.09,差异有统计学意义(P＜0.05).siRNA于扰PROM1表达能抑制U87细胞的生长和侵袭能力.转染PROM1siRNA组和对照组中穿膜细胞数分别为(85±9)个和(155±16)个,差异有统计学意义(P＜0.05).结论 miR-200b可通过靶向调控PROM1的表达而抑制胶质瘤细胞的侵袭力.
목적 탐토miR-200b파향조공PROM1적표체대효질류세포침습능력적영향이급miR-200b억류적분자궤제.방법 구건PROM1 3’단비번역구(3'UTR)형광소매보고재체,형광소매보고검측miR-200b대PROM1 3'UTR-형광소매활성적영향.장miR-200b모의물전염효질류U87세포,채용실시형광정량PCR화Western blot검측PROM1 mRNA화단백적표체수평.장PROM1 siRNA전염U87세포,Transwell침습실험검측PROM1하조대U87세포침습능력적영향.결과 쌍형광소매보고검측현시,miR-200b능특이성지여PROM1 3'UTR결합,억제기형광소매활성,기형광소매활성강도하강료57.0％,차이유통계학의의(P＜0.01).과표체miR-200b적U87세포중PROM1단백화mRNA표체수평균강저,전염miR-200b조화대조조중PROM1 mRNA적표체수평분별위0.64 ±0.05화0.95 ±0.09,차이유통계학의의(P＜0.05).siRNA우우PROM1표체능억제U87세포적생장화침습능력.전염PROM1siRNA조화대조조중천막세포수분별위(85±9)개화(155±16)개,차이유통계학의의(P＜0.05).결론 miR-200b가통과파향조공PROM1적표체이억제효질류세포적침습력.
Objective To explore whether miR-200b suppresses tumor cell invasion by targeting PROM1,thus to reveal the molecular mechanism that miR-200b functions as a tumor suppressor in glioma.Methods PROM I 3'UTR-luciferase vector was constructed and dual-luciferasc reporter gene assay was employed to examine the effect of miR-200b on luciferase activity.Human glioblastoma U87 cells were transfected with miR-200b mimics,and next qRT-PCR and Western blotting were performed to detect the expressions of PROM1 mRNA and protein.The effect of PROM1 down-regulation on invasion was observed after PROM1 siRNA were transfected into U87 cells.Results The miR-200b bound to the 3'-untranslated region (UTR) of PROM1 and inhibited the luciferase activity.Its luciferase activity was down-regulated by 57.0％ (P ＜0.01).PROM1 protein and mRNA expressions were significantly down-regulated when miR200b was overexpressed in the U87 cells (P ＜ 0.05).siRNA-mediated down-regulation of PROM1 suppressed the potential of cell invasion.The invasion ability of SKOV3 cells after transfection with siRNAPROM1 was significantly lower than that in the negative control cells (P ＜0.05).Conclusion miR-200b may suppress cell invasion by targeting PROM1 in glioma.