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钠氢交换蛋白1(sodium hydrogen exchange protein,NHE1)是调控细胞内酸碱平衡的膜蛋白。本研究旨在探究NHE1蛋白的表达对胶质母细胞瘤细胞迁移和侵袭影响。首先采用Matrigel-transwell侵袭实验比较胶质母细胞瘤U87和U251的侵袭能力,并分别分选出两细胞株强、弱亚群;采用反转录-实时荧光定量聚合酶链式反应(RT-q PCR)检测U87和U251细胞以及两细胞株强、弱亚群中NHE1 m RNA水平;然后采用靶向NHE1 m RNA的si RNA(si-NHE1)和si RNA阴性对照(si-NC)分别转染U87细胞,RT-q PCR法和蛋白质免疫印迹实验检测两组中NHE1 m RNA和蛋白水平;Transwell实验检测两组细胞的迁移和侵袭能力。结果显示U87细胞的侵袭能力强于U251细胞,NHE1 m RNA在U87细胞表达量是U251细胞中的50.08倍,NHE1 m RNA在两种细胞株的强侵袭性亚群中的表达量分别是弱侵袭性亚群的19.8倍和62.3倍,差异均具有统计学意义(p<0.05)。转染si-NHE1可有效降低U87细胞内NHE1的m RNA和蛋白水平。迁移实验中si-NHE1组和si-NC组细胞穿过8μm微孔滤膜数目分别是(188.33±16.04)和(138.00±9.54);侵袭实验中两组细胞穿过数目分别是(207.33±16.56)和(119.67±9.61),两实验数据差异均具有统计学意义(p<0.05)。以上研究表明,NHE1在胶质母细胞瘤中的表达呈异质性,其表达水平与胶质母细胞瘤细胞的侵袭能力能正相关。沉默NHE1可显著抑制胶质母细胞瘤细胞的迁移和侵袭能力。本研究为解析胶质母细胞瘤的侵袭性生长的特性和临床治疗胶质母细胞瘤提供了新的思路。
Abstract：The sodium hydrogen exchanger-1(NHE1) is a membrane protein that regulates intracellular acid-base balance. This study aimed to investigate the effects of NHE1 expression on the migration and invasion abilities of human glioblastoma cells. Matrigel-transwell assay was used to compare the invasiveness of glioblastoma cell lines U87 and U251, and to separate the strong and weak subgroups of them. Reverse transcription real-time fluorescent quantitative(RT-q PCR) was used to detect the level of NHE1 m RNA in U87, U251 and in their strong and weak subgroups. Then, si RNA targeting NHE1 m RNA(si-NHE1) and negative control of si RNA(si-NC) were transfected into U87 cells, respectively. RT-q PCR and western blot were used to identify the NHE1 m RNA and protein level of the two groups; transwell assay was performed to detect migration and invasion abilities of the two groups. The results showed that the NHE1 m RNA level in U87 cells was 50.08-fold as that in U251 cells. In addition, NHE1 m RNA levels in strong invasion subgroups of U87 and U251 cells were 19.81-fold and 62.33-fold as those in weak invasion subgroups, and the differences were statistically significant(p<0.05). The m RNA and protein levels of NHE1 were markedly down-regulated by transfected NHE1-si RNA. In the migration assay(without matrigel), the cell number invaded into the lower chamber in si-NHE1 and si-NC groups were(188.33±16.04) and(138.00±9.54), respectively. While the cell number in invasion assay(with matrigel) were(207.33±16.56) and(119.67±9.61), and the differences were statistically significant(p<0.05). These data indicated that NHE1 expression in glioblastoma is heterogeneous and its expression level is positively associated with the invasion ability of glioblastoma cells. The migration and invasion abilities of glioblastoma cells could be inhibited by silence of NHE1. This study provided new ideas for clarifying the molecular mechanisms of invasive growth and clinical treatment of glioblastoma.
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基本信息:
DOI：10.13417/j.gab.036.004984
中图分类号:R739.4
引用信息:
[1]石文健,赵喜庆,张欢,等.沉默NHE1抑制胶质母细胞瘤细胞的迁移和侵袭能力[J].基因组学与应用生物学,2017,36(12):4984-4991.DOI:10.13417/j.gab.036.004984.
基金信息:
河北省唐山市社会发展科研计划项目基金(14130258B)资助
2017-12-25
2017-12-25