干扰卷曲蛋白1基因表达抑制胶质瘤细胞增殖、迁移及上皮-间充质转化  

Impediment of frizzled-1 expression suppresses glioma cell proliferation, migration and epithelial-mesenchymal transition

作　　者：宋振宇[1] 刘献志[1] 刘增进[1] 吴力新[1] Song Zhenyu;Liu Xianzhi;Liu Zengjin;Wu Lixin(Department of Neurosurgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院神经外科,450052

出　　处：《中华实验外科杂志》2022年第7期1306-1309,共4页Chinese Journal of Experimental Surgery

摘　　要：目的观察卷曲蛋白1(FZD1)对神经胶质瘤细胞增殖、迁移和上皮-间充质转化的作用。方法将U118细胞分为低氧0、24、48 h组,利用实时定量聚合酶链反应(PCR)法和蛋白质印迹法(Western blot)法检测FZD1 mRNA和蛋白的表达。U118细胞分为对照Ⅰ组、对照短发卡RNA(shRNA)Ⅰ组、sh-缺氧诱导因子(HIF)-1α组、对照Ⅱ组、对照shRNAⅡ组、sh-FZD1组。利用脂质体法将对照短发卡RNA(Control shRNA)、HIF-1αshRNA、FZD1 shRNA转染胶质瘤细胞,噻唑蓝(MTT)法检测细胞增殖能力,平板克隆形成实验检测细胞克隆形成能力,小室迁移(Transwell)试验检测细胞迁移,Western blot检测HIF-1α、波形蛋白、N-钙黏蛋白、E-钙黏蛋白的表达。采用SPSS 19.0统计软件分析,多组间比较采用双因素方差分析或克鲁斯卡尔-沃利斯(Kruskal-Wallis)秩和检验。结果低氧24 h和48 h组细胞FZD1 mRNA和蛋白表达水平高于低氧0 h组(mRNA水平:2.46±0.39,3.54±0.44比1.00±0.04,F=83.537,P<0.01;蛋白水平:1.41±0.06,1.85±0.09比1.06±0.06,χ2=7.200,P<0.01)。sh-HIF-1α组HIF-1α和FZD1蛋白表达水平低于对照Ⅰ组和对照shRNAⅠ组(HIF-1α:0.50±0.06比1.03±0.03、0.97±0.03,χ2=7.200,P<0.01;FZD1:0.60±0.06比1.03±0.04、0.96±0.04,χ2=7.200,P<0.01)。培养24、48、72、96 h后,sh-FZD1组细胞活性低于对照Ⅱ组和对照shRNAⅡ组(24 h:0.159±0.006比0.204±0.008、0.216±0.008;48 h:0.218±0.009比0.329±0.009、0.331±0.009;72 h:0.296±0.007比0.447±0.011、0.472±0.013;96 h:0.361±0.008比0.563±0.013、0.582±0.015,F=32.790,P<0.01)。sh-FZD1组细胞克隆形成数量低于对照Ⅱ组和对照shRNAⅡ组(238.00±21.28比343.00±17.35、371.69±21.28,χ2=7.200,P<0.01)。sh-FZD1组细胞迁移数目低于对照Ⅱ组和对照shRNAⅡ组(18.33±2.52比31.33±3.51、36.67±2.08,χ2=6.880,P<0.01)。sh-FZD1组FZD1、波形蛋白、N-钙黏蛋白表达水平低于对照Ⅱ组和对照shRNAⅡ组,而E-钙黏蛋白表达高于对照Ⅱ组和对照shRNAⅡ组(FZD1:0.33±Objective To investigate the effect of frizzled-1(FZD1)on cell proliferation,migration and epithelial-mesenchymal transition of glioma.Methods U118 cells were randomly allocated into hypoxia 0,24 and 48 h group.The expression of FZD1 mRNA and protein was detected by quantitative real-time polymerase chain reaction(qPCR)and Western blotting.U118 cells were then divided into following groups:controlⅠ,control short harpin RNA(shRNA)Ⅰ,sh-hypoxia inducible factor(HIF)-1α,controlⅡ,control shRNAⅡ,and sh-FZD1.Control shRNA,HIF-1αshRNA,and FZD1 shRNA were transfected into glioma cells using liposome.Cell viabilities were evaluated by methyl thiazolyl tetrazolium(MTT)experiments,colony formation capabilities by plate colony formation assay,and cell migratory abilities by Transwell assay.Furthermore,Western blotting was used to detect the protein expression of HIF-1α,vimentin,N-cadherin,and E-cadherin.SPSS 19.0 software was employed for statistical analysis.Comparison among multiple groups was made with methods including analysis of variance or kruskal-Wallis rank sum test.When P value was less than 0.05,the difference was considered as significant.Results FZD1 mRNA and protein expression in hypoxia 24 h and 48 h groups was higher than in hypoxia 0 h group(mRNA level:2.46±0.39,3.54±0.44 vs.1.00±0.04,F=83.537,P<0.01;protein level:1.41±0.06,1.85±0.09 vs.1.06±0.06,χ2=7.200,P<0.01).Both HIF-1αand FZD1 proteins were decreased in sh-HIF-1αgroup as compared with controlⅠand control shRNAⅠgroups(HIF-1α:0.50±0.06 vs.1.03±0.03,0.97±0.03,χ2=7.200,P<0.01;FZD1:0.60±0.06 vs.1.03±0.04,0.96±0.04,χ2=7.200,P<0.01).After cultivation for 24,48,72 and 96 h,cell viabilities in sh-FZD1 group were inhibited and lower than controlⅡand control shRNAⅡgroups(24 h:0.159±0.006 vs.0.204±0.008,0.216±0.008;48 h:0.218±0.009 vs.0.329±0.009,0.331±0.009;72 h:0.296±0.007 vs.0.447±0.011,0.472±0.013;96 h:0.361±0.008 vs.0.563±0.013,0.582±0.015,F=32.790,P<0.01).Colony formation number in sh-FZD1 group was less than c

关 键 词：胶质瘤 卷曲蛋白1 增殖 迁移 上皮-间充质转化 

分 类 号：R739.4[医药卫生—肿瘤]