WDR12对神经胶质瘤细胞增殖、凋亡和周期的影响及其作用机制

Effect of WDR12 on proliferation,apoptosis and cell cycle of glioma cells and its mechanism

目的:探讨WDR12对神经胶质瘤细胞增殖、凋亡和周期的影响及其作用机制。方法:选取河南大学附属南阳市中心医院和河南中医药大学第一附属医院2017年6月到2019年6月收集的132例脑胶质瘤和癌旁组织作为研究对象,分析比较WDR12蛋白表达水平;人脑胶质瘤细胞系U251细胞分为对照组和WDR12 KD组,分别转染对照组短发卡RNA(shRNA)和WDR12 shRNA,48 h后采用细胞计数试剂盒(CCK-8)和克隆形成实验测定细胞增殖能力;采用流式细胞术分析两组细胞凋亡水平和周期变化;采用蛋白质印迹法(Western blot)分析WDR12对凋亡和周期相关蛋白的影响。组间数据比较采用t检验。结果:癌旁组织中WDR12蛋白相对表达水平(1.18±0.19)明显低于神经胶质瘤组织WDR12蛋白相对表达水平(3.18±0.29),差异有统计学意义(t=4.197,P<0.05)。对照组细胞培养48 h后吸光度(A)值(2.18±0.13)明显高于WDR12 KD组细胞48 h A值(1.53±0.21),差异有统计学意义(t=3.192,P<0.05)。对照组细胞克隆形成率[(79.43±9.32)%]明显高于WDR12 KD组细胞克隆形成率[(37.99±5.90)%],差异有统计学意义(t=5.119,P<0.05)。对照组细胞凋亡比例[(4.07±1.04)%]明显低于WDR12 KD组细胞凋亡比例[(15.88±3.90)%],差异有统计学意义(t=3.891,P<0.05)。对照组细胞S期比例[(36.19±4.25)%]明显高于WDR12 KD组细胞S期比例[(49.57±3.08)%],差异有统计学意义(t=3.215,P<0.05)。对照组细胞G 2期比例[(25.04±2.89)%]低于WDR12 KD组细胞G 2期比例[(17.53±4.29)%],差异有统计学意义(t=3.973,P<0.05)。对照组细胞半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3蛋白相对表达水平(1.07±0.10)明显低于WDR12 KD组细胞(2.76±0.12),差异有统计学意义(t=3.871,P<0.05)。对照组细胞CDK1蛋白相对表达水平(1.95±0.18)明显高于WDR12 KD组细胞(0.76±0.09),差异有统计学意义(t=3.011,P<0.05)。结论:WDR12在神经胶质瘤组织中呈高表达,敲降WDR12可抑制神经胶质瘤细胞增殖并诱导其凋亡和细胞周期阻滞。

Objective To investigate the effect of WDR12 on proliferation,apoptosis and cell cycle of glioma cells and its mechanism.Methods Totally,132 cases of glioma and adjacent tissues collected in Nanyang Central Hospital Affiliated to Henan University and the First Affiliated Hospital of Henan University of Traditional Chinese Medicine from June 2017 to June 2019 were selected as the research objects,and the expression level of WDR12 protein was analyzed and compared.Human glioma cell line U251 cells were divided into control group and WDR12 KD group.The control group and WDR12 group were transfected with short hairpin RNA(shRNA)and WDR12 shRNA respectively.After 48 h,cell proliferation was analyzed by cell counting kit-8(CCK-8)assay and clone formation assay.Cell apoptosis and cell cycle were analyzed by flow cytometry.The expression of apoptosis-and cycle-related proteins was analyzed by Western blotting.The data were compared between two groups by t-test.Results The relative expression level of WDR12 protein in adjacent tissues(1.18±0.19)was significantly lower than that in glioma tissue(3.18±0.29,t=4.197,P<0.05).The absorbance(A)value of the control group after 48 h of culture(2.18±0.13)was significantly higher than the WDR12 KD group(1.53±0.21,t=3.192,P<0.05).The colony formation rate in the control group[(79.43±9.32)%]was significantly higher than that in WDR12 KD group[(37.43±3.91)%,t=5.119,P<0.05].The apoptosis rate in control group[(4.07±1.04)%]was significantly lower than that in WDR12 KD group[(15.88±3.90)%,t=3.891,P<0.05].The proportion of S phase cells in the control group[(36.19±4.25)%]was significantly higher than that in WDR12 KD group[(49.57±3.08)%,t=3.215,P<0.05].The proportion of G2 phase cells in control group[(25.04±2.89)%]was significantly lower than that in WDR12 KD group[(17.53±4.29)%,t=3.973,P<0.05].The relative expression level of cysteinyl aspartate-specific protease(Caspase)-3 protein in the control group(1.07±0.10)was significantly lower than that in WDR12 KD group(2.76±0.12,t=3.871,P<0.05).The relative expression level of CDK1 protein in control group(1.95±0.18)was significantly higher than that in WDR12 KD group(0.76±0.09,t=3.011,P<0.05).Conclusion WDR12 is highly expressed in glioma tissues.Knockdown of WDR12 can inhibit the proliferation of glioma cells and induce apoptosis and cell cycle arrest.