敲低WTAP表达抑制脑胶质瘤干细胞的增殖、迁移和侵袭

Knockdown of WTAP expression inhibites proliferation,migration,and invasion of glioma stem cells

目的 探讨敲低Wilms肿瘤抑制因子(WTAP)表达对脑胶质瘤干细胞增殖、迁移和侵袭能力的影响及机制。方法 从手术切除胶质瘤标本中提取胶质瘤干细胞(GSC),应用lipofectamine2000转染WTAP shRNA质粒敲低WTAP表达,以shRNA为阴性对照;PCR检测WTAP mRNA水平分析敲低效率;MTT法检测细胞增殖能力,Transwell小室法检测细胞迁移和侵袭能力,免疫印迹法检测细胞AKT磷酸化水平。结果 从胶质瘤标本中成功提取GSC,其SOX2表达显著降低(P<0.001),而α-SMA和PDGFRb的表达上调(P<0.001)。与阴性对照组相比,WTAP敲低组GSC细胞WTAP mRNA水平明显降低(P<0.05),细胞增殖、迁移和侵袭能力均显著下降(P<0.05),细胞AKT的磷酸化水平显著降低(P<0.05)。结论 敲低WTAP表达可抑制GSC的增殖、迁移和侵袭能力,其机制可能与抑制AKT磷酸化有关。

Objective To investigate the effect of knockdown of Wilms tumor suppressor factor(WTAP)on proliferation,migration,and invasion of glioma stem cells and its mechanism.Methods Glioma stem cells(GSCs)were extracted from glioma specimens.WTAP shRNA plasmids were transfected into GSCs to knockdown the WTAP expression using lipofectamine2000 transfection kit.WTAP mRNA level was detected by PCR assay to analyze the efficiency of knockdown.GSCs proliferation was detected by MTT assay,migration and invasion abilities of GSCs were detected by Transwell assay,and AKT phosphorylation levels in GSCs were detected by western blotting.Results Tumor cells extracted from glioma specimens were cultured in GSC medium and showed high expression of the stem cell marker SOX2.When EGF and FGF2 were replaced with TGFβin the medium,the expression of stem cell marker SOX2 was significantly decreased(P<0.001),while the expression of peritascular cell markersα-SMA and PDGFRb were significantly up-regulated(P<0.001).Compared with negative control group,proliferation,migration,and invasion were significantly inhibited(P<0.05),and the phosphorylation level of AKT was significantly decreased(P<0.05)in GSCs with knockdown of WTAP expression.Conclusions Knockdown of WTAP expression can inhibit the proliferation,migration,and invasion of GSCs,which may be related to the inhibition of AKT phosphorylation.