WW域结合蛋白2对脑胶质瘤细胞增殖、迁移和代谢的影响

Effects of WW domain binding protein 2 on proliferation,migration and metabolism of glioma cells

目的探讨WW域结合蛋白2(WBP2)对脑胶质瘤细胞增殖、迁移和代谢的影响及其机制。方法2015年6月到2018年6月安阳市人民医院收集正常脑胶质和脑胶质瘤组织59例,采用免疫组织化学分析脑胶质和胶质瘤组织中WBP2蛋白的表达水平。采用WBP2短发卡RNA(shRNA)和对照shRNA慢病毒感染U251脑胶质瘤细胞株,建立WBP2沉默和对照细胞株,分别为实验组和对照组。采用细胞计数试剂盒(CCK-8)试剂盒和5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色分析两组细胞的增殖能力;采用Transwell分析两组细胞的迁移能力;采用异种移植瘤模型分析两组细胞在体内的增殖能力。采用乳酸试剂盒分析两组细胞乳酸分泌。采用蛋白质印迹法(Western blot)分析糖酵解甘油醛-3-磷酸脱氢酶(GAPDH)和乳酸脱氢酶(LDH)的表达。采用t检验。结果与正常脑胶质细胞(54.31±5.69)比较,脑胶质瘤组织中WBP2蛋白表达水平(169.57±10.69)显著升高,差异有统计学意义(t=4.312,P<0.01)。与对照组细胞(1.59±0.26)比较,实验组细胞72 h增殖能力(1.05±0.15)明显下降,差异有统计学意义(t=3.105,P<0.05)。与对照组细胞EdU染色率[(67.25±6.98)%]比较,实验组EdU染色率[(19.33±4.89)%]显著下降,差异有统计学意义(t=2.954,P<0.05)。实验组细胞迁移数量[(64.32±8.91)个]较对照组[(21.38±11.20)个]明显下降,差异有统计学意义(t=3.984,P<0.01)。实验组细胞培养上清乳酸[(68.54±6.87)μmol/L]含量较对照组[(100.85±9.51)μmol/L]显著下调,差异有统计学意义(t=3.029,P<0.05)。结论WBP2蛋白在脑胶质瘤组织中呈高表达,参与胶质瘤细胞的增殖和迁移,并可调节肿瘤细胞糖酵解。

Objective To investigate the effect of WW domain binding protein 2(WBP2)on proliferation,migration and metabolism of glioma cells and the molecular mechanism.Methods Fifty-nine cases of normal gliomas and gliomas collected in our hospital from June 2015 to June 2018 were studied.The expression level of WBP2 protein in glial tissues and gliomas tissues was analyzed by immunohistochemistry.WBP2 short hairpin RNA(shRNA)and control shRNA lentivirus were used to infect U251 glioma cell lines to establish WBP2 silencing and control cell lines.The proliferation ability of the two groups was analyzed by cell counting kit-8(CCK-8)and 5-Ethynyl-2’-deoxyuridine(EdU)staining.The migration ability of the two groups was analyzed by Transwell.The tumor growth of the two groups in vivo was analyzed by anaxenograft tumor model.Lactate secretion was analyzed by lactate kit.The expression of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and lactate dehydrogenase(LDH)in glycolysis pathways was analyzed by Western blotting.Results The expression level of WBP2 protein in glioma cancer tissues was significantly higher than that in normal brain glial cells[(169.57±10.69)vs.(54.31±5.69)](t=4.312,P<0.01).Compared with the control group(1.59±0.26),the cell proliferation ability in the experimental group(1.05±0.15)significantly decreased at 72 h(t=3.105,P<0.05).Compared with the control group[(67.25±6.98)%],the EdU staining rate in the experimental group[(19.33±4.89)%]significantly decreased(t=2.954,P<0.05).The number of cell migration in the experimental group[(64.32±8.91)cells]was significantly less than that in the control group[(21.38±11.20)cells,t=3.984,P<0.01].The content of lactic acid[(68.54±6.87)μmol/L]in cell culture supernatant of experimental group was significantly lower than that of control group[(100.85±9.51)μmol/L,t=3.029,P<0.05].Conclusion WBP2 protein is highly expressed in gliomas,which may participate in the proliferation and migration of gliomas and regulate the glycolysis of gliomas.