脂肪非典型钙黏蛋白-1靶向调控缺氧诱导因子-1α对高级别胶质瘤侵袭的研究

Fat atypical cadherin 1 regulating invasion by targeting hypoxia inducible factor-1α in high grade glioma

目的探讨脂肪非典型钙黏蛋白（FAT1）通过靶向调控缺氧诱导因子-1α（HIF-1α）调节缺氧条件下胶质瘤细胞侵袭能力的作用机制。 方法选择我院收治的48例Ⅲ～Ⅳ级胶质瘤患者的手术病理组织样本，采用实时荧光定量聚合酶链反应（FQ-PCR）技术检测FAT1和HIF-1α表达水平并分析其相关性。以人胶质瘤细胞U87MG和GOS3作为研究对象，采用FQ-PCR技术检测正常氧气和缺氧条件下FAT1和HIF-1α表达。采用脂质体2000法将FAT1小干扰RNA（siRNA）、HIF-1α siRNA以及对照siRNA转染至U87MG和GOS3细胞株，检测正常氧气和缺氧条件下FAT1和HIF-1α表达，并采用Transwell小室技术检测U87MG和GOS3胶质瘤细胞的侵袭能力。 结果48例Ⅲ～Ⅳ级胶质瘤患者病理组织FAT1和HIF-1α相对表达水平分别为4.07（0.72，13.42）和0.94（0.29，3.49），Spearman相关分析显示FAT1和HIF-1α相对表达水平明显相关（r＝0.835，P＝0.000）。正常氧气浓度下GOS3细胞株FAT1（0.29±0.03）和HIF-1α（0.34±0.09）相对表达水平均显著低于U87MG细胞株的1.08±0.15、1.00±0.12，差异有统计学意义（t＝8.945、7.621，P＝0.010、0.002）；缺氧条件下，U87MG和GOS3细胞株FAT1和HIF-1α相对表达水平均显著高于正常氧气浓度下（t＝17.994、8.920、14.047、4.037，P＝0.000、0.001、0.004、0.016）；U87MG细胞株缺氧条件下FAT1（4.38±0.28）和HIF-1α（2.76±0.32）相对表达水平均显著高于GOS3细胞株的1.69±0.17、0.83±0.19，差异有统计学意义（t＝14.224、8.982，P＝0.000、0.001）。转染FAT1 siRNA后U87MG和GOS3细胞在正常氧气和缺氧条件下FAT1（0.17±0.06、0.16±0.03、0.16±0.03、0.18±0.03）和HIF-1α（0.18±0.03、0.17±0.04、0.15±0.04、0.21±0.02）相对表达水平均显著降低，差异均有统计学意义（t＝7.577、7.793、7.793、7.783、9.285、9.260、9.484、9.042，P＝0.002、0.013、0.013、0.014、0.009、0.007、0.007、0.011）；转染HIF-1α siRNA后U87MG和GOS3细胞在正常氧条件下的FAT1（0.16±0.04、0.28±0.03）以及正常氧气和缺氧条件下HIF-1α（0.15±0.03、0.16±0.02、0.15±0.03、0.17±0.02）相对表达水平显著降低，差异均有统计学意义（t＝7.890、6.834、9.624、9.614、9.624、9.500，P＝0.012、0.018、0.008、0.009、0.008、0.010）；但缺氧条件下的FAT1（4.27±0.41、2.72±0.25）相对表达水平显著高于正常氧，差异均有统计学意义（t＝12.649、9.671，P＝0.000、0.001）。缺氧条件下，转染FAT1 siRNA、转染HIF1α siRNA的U87MG细胞侵袭细胞数量[（23.2±4.2）、（21.5±3.1）个]显著低于正常氧条件[（57.6±8.1）、（58.7±7.6）个]，差异有统计学意义（t＝6.530、7.850，P＝0.003、0.001）。 结论高级别胶质瘤中FAT1和HIF-1α表达具有较强的相关性，缺氧条件下两者高表达，FAT1可通过靶向调控HIF-1α促进胶质瘤细胞侵袭。

ObjectiveTo investigate the action mechanism of fat atypical cadherin 1 （FAT1） regulating invasion by targeting hypoxia inducible factor-1α （HIF-1α） in high grade glioma under hypoxia. MethodsThe expression levels of FAT1 and HIF-1α in 48 cases of grade Ⅲ-Ⅳ glioma athological tissue samples in our hospital were detected by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）. The relationship between the expression of FAT1 and HIF-1α was analyzed. The expression of FAT1 and HIF-1α in human glioma cells U87MG and GOS3 under the condition of normal oxygen （20% O2） and hypoxia （1% O2） was detected by FQ-PCR. FAT1 small interfering RNA （siRNA）, HIF-1α siRNA and control siRNA were transfected into U87MG and GOS3 cell lines respectively by Lipofectamine 2000. The expression of FAT1 and HIF-1 under the normal oxygen and hypoxia conditions was detected. The invasive abilities of U87MG and GOS3 glioma cells were examined by cell chamber technique. ResultsThe relative expression levels of FAT1 and HIF-1α in 48 cases of pathological tissues of patients with grade Ⅲ-Ⅳ glioma were 4.07 （0.72, 13.42） and 0.94 （0.29, 3.49） respectively. Spearman analysis showed that there was significant correlation between the relative expression levels of FAT1 and HIF-1α （r=0.835, P=0.000）. The normal oxygen concentration in GOS3 cell lines FAT1 （0.29±0.03） and HIF-1α （0.34±0.09） relative expression levels were significantly lower than that of U87MG cells （1.08±0.15, 1.00±0.12; t=8.945, 7.621; P=0.010, 0.002）; hypoxia condition, U87MG and GOS3 cell lines FAT1 and HIF-1α relative expression levels were significantly higher than the normal oxygen concentration （t=17.994, 8.920, 14.047, 4.037; P=0.000, 0.001, 0.004, 0.016）; U87MG cells under hypoxic conditions FAT1 （4.38±0.28） and HIF-1α （2.76±0.32） relative expression levels were significantly higher than that of GOS3 cell line （1.69±0.17, 0.83±0.19; t=14.224, 8.982; P=0.000, 0.001）. After transfection of FAT1 siRNA, FAT1 （0.17±0.06, 0.16±0.03, 0.16±0.03, 0.18±0.03） and HIF-1α （0.18±0.03, 0.17±0.04, 0.15±0.04, 0.21±0.02） relative expression levels of U87MG and GOS3 cells in the normal oxygen and hypoxia were significantly decreased （t=7.577, 7.793, 7.793, 7.783, 9.285, 9.260, 9.484, 9.042; P=0.002, 0.013, 0.013, 0.014, 0.009, 0.007, 0.007, 0.011）. After transfection of HIF-1α siRNA, FAT1 （0.16±0.04, 0.28±0.03） relative expression levels in the normal oxygen and HIF-1α （0.15±0.03, 0.16±0.02, 0.15±0.03, 0.17±0.02） relative expression levels of U87MG and GOS3 cells in normal oxygen and hypoxia conditions were significantly decreased （t=7.890, 6.834, 9.624, 9.614, 9.624, 9.500; P=0.012, 0.018, 0.008, 0.009, 0.008, 0.010）; but the level under hypoxic conditions relative expression of FAT1 （4.27±0.41, 2.72±0.25） were significantly higher than that in normal oxygen conditions （t=12.649, 9.671; P=0.000, 0.001）. Under hypoxic conditions, the invasion cell quantity of U87MG cells transfected with FAT1 siRNA and HIF-1α siRNA [（23.2±4.2） and （21.5±3.1） cells] were lower than those of normal oxygen conditions [（57.6±8.1） and （58.7±7.6） cells; t=6.530, 7.850; P=0.003, 0.001]. ConclusionThe expression of FAT1 and HIF-1 in high grade gliomas is strongly correlated, and the expression is high under the hypoxia condition. FAT1 can promote the invasion of glioma cells by targeting HIF-1α.