CIB1通过Wnt/β-catenin信号通路调控三阴性乳腺癌细胞增殖和迁移及侵袭机制研究

Regulation of CIB1on the proliferation,migration and invasion of triple negative breast cancer cells through Wnt/β-catenin signaling pathway

目的探讨CIB1对三阴性乳腺癌MDA-MB-231和MDA-MB-468细胞增殖、迁移和侵袭的影响及作用机制。方法通过慢病毒转染构建过表达CIB1的MDA-MB-231和MDA-MB-468细胞,实验分为NC组和CIB1组。采用细胞计数试剂盒8(CCK8)、5-乙炔基-2′脱氧尿嘧啶核苷(EdU)实验检测细胞增殖活性,流式细胞术检测细胞凋亡情况,划痕实验和Transwell实验检测细胞迁移及侵袭能力。同时,采用实时荧光定量-聚合酶链式反应(qRT-PCR)和蛋白质印迹实验检测细胞内β-catenin、活化蛋白C(APC)、糖原合酶激酶3β(GSK-3β)、c-myc的mRNA和蛋白表达情况。结果MDA-MB-231细胞内NC组和CIB1组CIB1蛋白表达水平分别为0.75±0.17和1.53±0.38,t=3.267,P=0.031。MDA-MB-468细胞内NC组和CIB1组CIB1蛋白表达水平分别为0.91±0.01和1.12±0.07,t=4.953,P=0.008。CCK8实验结果显示,MDA-MB-231细胞中NC组和CIB1组增殖力差异有统计学意义,F_(组别)=120.088,P_(组别)<0.001;F_(时间)=408.784,P_(时间)<0.001;F_(组别×时间)=14.177,P_(组别×时间)<0.001。MDA-MB-468细胞中NC组和CIB1组增殖力差异有统计学意义,F_(组别)=277.649,P_(组别)<0.001;F_(时间)=1281.882,P_(时间)<0.001;F_(组别×时间)=33.378,P_(组别×时间)<0.001。EdU实验结果显示,MDA-MB-231细胞中NC组和CIB1组增殖率分别为(31.42±0.01)%和(46.20±0.02)%,t=14.610,P<0.001;MDA-MB-468细胞中NC组和CIB1组增殖率分别为(30.57±0.02)%和(42.61±0.03)%,t=6.397,P=0.003。MDA-MB-231细胞中NC组和CIB1组细胞凋亡率分别为(4.77±0.01)%和(2.07±0.10)%,t=3.785,P=0.019;MDA-MB-468细胞中NC组和CIB1组细胞凋亡率分别为(9.53±0.00)%、(3.90±0.00)%,t=19.390,P<0.001。MDA-MB-231细胞中NC组和CIB1组细胞迁移率分别为(46.20±0.01)%和(64.35±0.10)%,t=3.386,P=0.028;MDA-MB-468细胞中NC组和CIB1组细胞迁移率分别为(24.84±0.05)%和(56.83±0.06)%,t=7.278,P=0.002。MDA-MB-231细胞中NC组和CIB1组穿膜细胞数分别为(300.67±25.17)和(411.67±24.99)个,t=5.421,P=0.006;MDA-MB-468细胞中NC组和CIB1组穿膜细胞数分别为(257.00±31.43)和(388.00±8.72)个,t=6.956,P=0.002。qRT-PCR和蛋白质印迹实验结果显示,MDA-MB-231和MDA-MB-468细胞中CIB1组β-catenin、APC、c-myc mRNA和蛋白表达均升高,GSK-3βmRNA和蛋白表达均降低,差异均有统计学意义,均P<0.05。结论CIB1促进乳腺癌细胞增殖、侵袭和迁移能力,抑制细胞凋亡,可能通过促进Wnt/β-catenin信号通路的激活发挥促癌作用。

Objective To investigate the effect and mechanism of CIB1 on the proliferation,migration and invasion of MDA-MB-231 and MDA-MB-468 cells in triple negative breast cancer.Method MDA-MB-231 and MDA-MB-468 cells over expressing CIB1 were constructed by lentiviral transfection,and the experiment was divided into NC group and CIB1 group.Cell proliferation activity was detected using cell count kit 8(CCK8)and 5-ethynyl-2'deoxyuridine nucleoside(EdU)assay,flow cytometry was used to detect cell apoptosis,and scratch and transwell assays was used to detect cell migration and invasion ability.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and western blotting experiments were used to detect mRNA and protein expression of intracellularβ-catenin,activated protein C(APC),and glycogen synthase kinase 3β(GSK-3β)and c-myc.Results The expression levels of CIB1 protein in NC group and CIB1 group of MDAMB-231 cells were 0.75±0.17 and 1.53±0.38,t=3.267,P=0.031.The expression levels of CIB1 protein in NC group and CIB1 group of MDA-MB-468 cells were 0.91±0.01 and 1.12±0.07,t=4.953,P=0.008.The CCK8 experiment results showed that there was a statistically significant difference in proliferation ability between NC group and CIB1 group in MDA-MB-231 cells,F_(group)=120.088,P_(group)<0.001;F_(time)=408.784,P_(time)<0.001;F_(group×time)=14.177,P_(group×time)<0.001.There was a statistically significant difference in proliferation ability between NC group and CIB1 group in MDAMB-468 cells,F_(group)=277.649,P_(group)<0.001;F_(time)=1281.882,P_(time)<0.001;F_(group×time)=33.378,P_(group×time)<0.001.The EdU experiment results showed that,the proliferation rates of NC group and CIB1 group in MDA-MB-231 cells were(31.42±0.01)%and(46.20±0.02)%,t=14.610,P<0.001;The proliferation rates of NC group and CIB1 group in MDA-MB-468 cells were(30.57±0.02)%and(42.61±0.03)%,t=6.397,P=0.003.The apoptosis rates of NC group and CIB1 group in MDA-MB-231 cells were(4.77±0.01)%and(2.07±0.10)%,t=3.785,P=0.019;The apoptosis rates of NC group and CIB1 group in MDA-MB-468 cells were(9.53±0.00)%and(3.90±0.00)%,t=19.390,P<0.001.The cell migration rates of NC group and CIB1 group in MDA-MB-231 cells were(46.20±0.01)%and(64.35±0.10)%,t=3.386,P=0.028;The migration rates of NC group and CIB1 group in MDA-MB-468 cells were(24.84±0.05)%and(56.83±0.06)%,t=7.278,P=0.002.The number of transmembrane penetrating cells in NC group and CIB1 group of MDA-MB-231 cells were 300.67±25.17 and 411.67±24.99,t=5.421,P=0.006;The number of transmembrane penetrating cells in NC group and CIB1 group of MDA-MB-468 cells were 257.00±31.43 and 388.00±8.72,t=6.956,P=0.002.qRT-PCR and western blot experiments showed that the expressions ofβ-catenin,APC,c-myc mRNA and protein in CIB1 group were increased,mRNA and protein expressions of GSK-3βwere decreased in MDA-MB-231and MDA-MB-468 cells,all P<0.05.Conclusions CIB1 may promote the proliferation,invasion and migration of breast cancer cells,and inhibit the apoptosis of breast cancer cells through Wnt/β-catenin signaling pathway to promote breast cancer progression.