Hax-1对胶质瘤细胞增殖、迁移和侵袭的作用

目的 探讨造血干细胞特异性相关结合蛋白-1（Hax-1）对胶质瘤细胞增殖、迁移和侵袭的影响。方法 选择2018年9月至2019年6月手术切除并得到术后病理证实的胶质瘤组织35例和颅脑损伤内减压术切除的正常脑组织35例，采用qRT-PCR检测Hax-1 mRNA水平；同时检测胶质瘤细胞系（U87、A172、T98及U343）和人星形胶质细胞（NHAs）Hax-1 mRAN水平。使用Lipofectamine 2000法将Hax-1 siRNAs和阴性对照siRNA转染U87细胞构建Hax-1低表达细胞株，将Hax-1高表达质粒和PCDNA3.1空载质粒转染U343细胞构建Hax-1过表达细胞株，使用蛋白印迹法验证转染效果，采用CCK-8法检测细胞增殖，采用Transwell小室实验检测细胞迁移和侵袭，免疫印迹法检测NF-κB信号通路相关蛋白表达水平（NF-κB、CCND1、C-myc、MMP-2、MMP-9）。结果 胶质瘤组织Hax-1 mRNA水平明显高于正常脑组织（P<0.05）；胶质瘤细胞系（U87、A172、T98及U343）Hax-1 mRNA水平明显高于人星形胶质细胞（P<0.05），其中U87细胞Hax-1 mRNA水平最高，U343细胞最低。U343细胞转染Hax-1过表达质粒后，Hax-1蛋白水平显著增高（P<0.05），细胞增殖、侵袭、迁移能力均明显增强（P<0.05），NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显增高（P<0.05）。U87细胞转染Hax-1 siRNA后Hax-1蛋白水平显著降低（P<0.05），细胞增殖、侵袭、迁移能力均明显降低（P<0.05），NF-κB p65及IκBα蛋白磷酸化水平以及CCND1、C-Myc、MMP-2、MMP-9蛋白表达水平明显降低（P<0.05）。结论 胶质瘤组织Hax-1呈高表达，明显促进肿瘤细胞增殖、侵袭和迁移，其机制可能与激活NF-κB信号通路有关。

Effect of Hax-1 on proliferation,migration and invasion of glioma cells

Objective To explore the effect of hematopoietic stem cell-specific binding protein-1(Hax-1)on the proliferation,migration and invasion of glioma cells.Methods The mRNA levels of Hax-1 were detected by qRT-PCR in glioma tissues obtained from 35 patients with glioma who underwent surgery and in normal brain tissues obtained from 35 patients with traumatic brain injury who underwent decompression from September 2018 to June 2019,and in cultured cells including glioma cell lines(U87,A172,T98 and U343)and human astrocytes(NHAs).The cell line with low-expression of Hax-1 was constructed using Lipofectamine 2000 method to transfect Hax-1 siRNAs and negative control siRNA into U87 cells,and the cell line with high-expression of Hax-1 was constructed using Lipofectamine 2000 method to transfect Hax-1 high-expression plasmid and PCDNA3.1 empty plasmid into U343 cells,and Western blotting was used to verify the transfection efficiency,CCK-8 method was used to detect the cell proliferation,Transwell chamber experiment was used to detect the cell migration and invasion,and Western blotting was used to detect the expression levels of NF-κB signaling pathway related proteins(NF-κB,CCND1,C-myc,MMP-2,MMP-9).Results The level of Hax-1 mRNA in glioma tissue was significantly higher than that of normal brain tissue(P<0.05).The levels of Hax-1 mRNA in glioma cell lines(U87,A172,T98 and U343)were significantly higher than that of human astrocytes(P<0.05).Among glioma cell lines,the level of Hax-1 mRNA was the highest in U87 cells and the lowest in U343 cells.Up-regulation of Hax-1 expression in U343 cells significantly promoted cell proliferation,invasion,and migration(P<0.05),and increased the expression levels of phosphorylation of NF-κB p65 and IκBα,CCND1,C-Myc,MMP-2 and MMP-9(P<0.05).Down-regulation of Hax-1 expression in U87 cells significantly inhibited cell proliferation,invasion,and migration(P<0.05),and decreased the expression levels of phosphorylation of NF-κB p65 and IκBα,CCND1,C-Myc,MMP-2 and MMP-9(P<0.05).Conclusions Hax-1 is highly expressed in glioma tissues,which significantly promotes tumor cell proliferation,invasion and migration.The mechanism may be related to the activation of NF-κB signaling pathway.