CAJ | 학술논문

目的:观察抑制恶性胶质瘤LN229细胞中Girdin蛋白的表达对细胞运动的影响,并探讨其分子机制.方法:用小RNA干扰技术降低恶性胶质瘤细胞株LN229中Girdin蛋白的表达水平,通过Western blotting技术验证其降表达效率,并筛选出稳定降表达的克隆命名为siGirdin/LN229,阴性对照组命名为scr/LN229.利用增殖实验检测LN229细胞的存活率;应用划痕实验和侵袭实验检测细胞的运动变化.通过慢病毒感染技术来提高Girdin在LN229细胞中的表达水平,使用嘌呤霉素筛选2周建立稳定的细胞系,并用Western blot验证Girdin表达水平.结果:Western blotting验证小RNA干扰技术有效沉默了LN229细胞中Girdin的表达.增殖实验显示siGirdin/LN229细胞增殖能力下降(P<0.05),至第6天时实验组细胞数为(19.98±2.54)×104;对照组细胞数为(61.04±5.26)×104.划痕实验发现细胞非定向运动能力减弱(P<0.05),24 h时实验组细胞运动的距离为(21.83±2.52)μm;对照组为(44.16±2.89)μm.Western blotting结果表明LN229细胞中Girdin的表达增高,增殖实验显示过表达Girdin对LN229细胞增殖能力无影响.体外细胞侵袭实验中实验组穿过Matrigel胶的细胞数为(14.67±2.08)个;对照组为(32.67±3.06)个,结果显示 Girdin降表达后细胞的侵袭能力较对照组细胞显著下降(P<0.05).结论:Girdin和胶质瘤细胞增殖和运动能力密切相关,Girdin蛋白表达降低可抑制LN229细胞的增殖、迁移和侵袭能力.
목적:관찰억제악성효질류LN229세포중Girdin단백적표체대세포운동적영향,병탐토기분자궤제.방법:용소RNA간우기술강저악성효질류세포주LN229중Girdin단백적표체수평,통과Western blotting기술험증기강표체효솔,병사선출은정강표체적극륭명명위siGirdin/LN229,음성대조조명명위scr/LN229.이용증식실험검측LN229세포적존활솔;응용화흔실험화침습실험검측세포적운동변화.통과만병독감염기술래제고Girdin재LN229세포중적표체수평,사용표령매소사선2주건립은정적세포계,병용Western blot험증Girdin표체수평.결과:Western blotting험증소RNA간우기술유효침묵료LN229세포중Girdin적표체.증식실험현시siGirdin/LN229세포증식능력하강(P<0.05),지제6천시실험조세포수위(19.98±2.54)×104;대조조세포수위(61.04±5.26)×104.화흔실험발현세포비정향운동능력감약(P<0.05),24 h시실험조세포운동적거리위(21.83±2.52)μm;대조조위(44.16±2.89)μm.Western blotting결과표명LN229세포중Girdin적표체증고,증식실험현시과표체Girdin대LN229세포증식능력무영향.체외세포침습실험중실험조천과Matrigel효적세포수위(14.67±2.08)개;대조조위(32.67±3.06)개,결과현시 Girdin강표체후세포적침습능력교대조조세포현저하강(P<0.05).결론:Girdin화효질류세포증식화운동능력밀절상관,Girdin단백표체강저가억제LN229세포적증식、천이화침습능력.
Central Laboratory of Oncology, 2Department of Breast Pathology, Tianjin Medical University Cancer Institute and Hospital; Key Laboratory of Breast Cancer Prevention and Treatment of the Ministry of Education; Key Laboratory of Cancer Prevention and Treatment of Tianjin, Tianjin 300060, China @@@@ Objective: The objectives of this study were to investigate the role of Girdin protein in the motility of LN229 glioblastoma cells using small RNA interference technology and to determine the underlying molecular mechanisms involved. Methods: The expression of Girdin was knocked down in LN229 glioblastoma cells by Girdin siRNA. It was identified by Western blot analysis. Stable clones that could detect the cell survival ratio were used in the proliferation assay. Scratch and invasion in vitro assays were also used to investigate the motility of the cells. Results: Western blot analysis showed that RNA interference significantly decreased the expression level of Girdin. Proliferation assay revealed that the number of siGirdin/LN229 cells decreased compared with that of scr/LN229 cells. On the 6th day, the number of siGirdin/LN229 cells was 19.98 ± 2.54 × 104, whereas that of scr/LN229 cells was 61.04 ± 5.26 × 104. At 24 h after wounding, the migrated distances of the siGirdin/LN229 and scr/LN229 cells were 21.83 ± 2.52 and 44.16 ± 2.89 μm, respectively. Moreover, although the expression level of Girdin increased, the number of PCDH-p-Girdin/LN229 cells did not increase compared with that of PCDH-p-vector/LN229 cells. Invasion in vitro assay demonstrated that the numbers of siGirdin/LN229 and scr/LN229 transmembrane cells were 14.67 ± 2.08 and 32.67 ± 3.06, respectively. Conclusion: Girdin is essential to the proliferation and motility of glioblastoma cells.