1,4,5-三磷酸肌醇3激酶A调控脑胶质瘤细胞增殖、迁移和上皮间质化

Modulation of glioma cell proliferation,migration and epithelial-mesenchymal transition by inositol-1,4,5-trisphosphate 3-kinase-A

目的探讨1,4,5-三磷酸肌醇3激酶A(ITPKA)在脑胶质瘤细胞的表达和功能。方法利用实时定量聚合酶链反应(PCR)和蛋白质印迹法(Western blot)比较SVGP12、U118和U87细胞系(购自中国科学院生物化学与细胞生物学研究所)ITPKA mRNA和蛋白表达水平。将U87细胞分为对照组、对照短发卡RNA(shRNA)组和sh-ITPKA组,噻唑蓝法检测转染细胞培养24、48、72、96 h后细胞活性,平板克隆和小室迁移(Transwell)法检测克隆形成能力与细胞迁移能力,Western blot检测ITPKA、波形蛋白、N-钙黏蛋白和E-钙黏蛋白表达水平。多组间比较采用单因素方差分析或双因素方差分析。结果U118和U87细胞中ITPKA mRNA和蛋白表达水平明显高于SVGP12细胞(mRNA水平:2.81±0.40、3.92±0.19比1.06±0.06,F=94.48,P<0.01;蛋白水平:0.57±0.05、0.66±0.03比0.20±0.01,F=161.30,P<0.01)。培养24、48、72、96 h后,sh-ITPKA组细胞活性明显低于对照组和对照shRNA组(24 h:0.14±0.01比0.19±0.01、0.20±0.02,P<0.01;48 h:0.20±0.01比0.30±0.01、0.30±0.02,P<0.01;72 h:0.26±0.01比0.37±0.01、0.38±0.01,P<0.01;96 h:0.29±0.01比0.45±0.01、0.45±0.01,F_(交互)=17.22,F_(时间)=713.00,F_(分组)=662.60,P<0.01)。sh-ITPKA组细胞细胞迁移数目低于对照组和对照shRNA组(25.40±2.51比41.20±4.66、42.00±5.24,F=23.69,P<0.01)。同时,sh-ITPKA组细胞克隆形成数目明显低于对照组和对照shRNA组(120.60±11.41比215.00±18.73、221.40±14.83,F=68.15,P<0.01)。sh-ITPKA组细胞ITPKA、波形蛋白、N-钙粘蛋白表达水平低于对照组和对照shRNA组,而E-钙粘蛋白表达则高于对照组和对照shRNA组(ITPKA:0.11±0.02比0.70±0.02、0.68±0.03,F=637.00,P<0.01;波形蛋白:1.06±0.07比1.65±0.07、1.60±0.08,F=58.31,P<0.01;N-钙粘蛋白:0.88±0.04比1.15±0.07、1.20±0.03,F=31.56,P<0.01;E-钙粘蛋白:0.85±0.03比0.46±0.04、0.43±0.04,F=116.80,P<0.01)。结论ITPKA在胶质瘤细胞中表达升高,敲低ITPKA表达则抑制胶质瘤细胞恶性行为。

Objective To investigate the expression and function of inositol-1,4,5-trisphosphate 3-kinase-A(ITPKA)in glioma cells.Methods The expression of ITPKA mRNA and protein in SVGP12,U118 and U87 cell lines(provided by cell bank of chinese academy of sciences)were analyzed by quantitative Real-time polymerase chain reaction(qPCR)and Western blotting,respectively.U87 cells were randomly allocated into control,control short hairpin RNA(shRNA)and sh-ITPKA group.Cell viabilities were assayed using methyl thiazolyl tetrazolium(MTT)method.Colony formation capabilities and cell migration by plate colony formation assay and transwell assay.Protein expression of ITPKA,vimentin,N-cadherin and E-cadherin was detected using Western blotting.Comparison among multiple groups was made with methods including one-way or two-way analysis of variance(ANOVA).Results Compared with SVGP12 cells,the expression of ITPKA mRNA and protein was upregulated in U118 and U87 cells(mRNA level:2.81±0.40,3.92±0.19 vs.1.06±0.06,F=94.48,P<0.01;protein level:0.57±0.05,0.66±0.03 vs.0.20±0.01,F=161.30,P<0.01).Following culturing for 24,48,72 and 96 h,compared with control and control shRNA group,cell viabilities of sh-ITPKA group were significantly reduced(24 h:0.14±0.01 vs.0.19±0.01,0.20±0.02,P<0.01;48 h:0.20±0.01 vs.0.30±0.01,0.30±0.02,P<0.01;72 h:0.26±0.01 vs.0.37±0.01,0.38±0.01,P<0.01;96 h:0.29±0.01 vs.0.45±0.01,0.45±0.01,F_(interaction)=17.22,F_(time)=713.00,F_(group)=662.60,P<0.01).Migratory cell numbers were diminished in sh-ITPKA group compared with control and control shRNA group(25.40±2.51 vs.41.20±4.66,42.00±5.24,F=23.69,P<0.01).Meanwhile,colony formation numbers in sh-ITPKA group were decreased when compared with control and control shRNA group(120.60±11.41 vs.215.00±18.73,221.40±14.83,F=68.15,P<0.01).Additionally,the expression of ITPKA,vimentin,and N-cadherin was downregulated while E-cadherin was upregulated in ITPKA group compared with control and control shRNA group(ITPKA:0.11±0.02 vs.0.70±0.02,0.68±0.03,F=637.00,P<0.01;vimentin:1.06±0.07 vs.1.65±0.07,1.60±0.08,F=58.31,P<0.01;N-cadherin:0.88±0.04 vs.1.15±0.07,1.20±0.03,F=31.56,P<0.01;E-cadherin:0.85±0.03 vs.0.46±0.04,0.43±0.04,F=116.80,P<0.01).Conclusion ITPKA expression was upregulated in glioma.Knockdown of ITPKA suppressed malignant behaviors of glioma cells.