CENP-A通过调控PI3K/AKT/NF-κB信号通路对卵巢癌细胞侵袭、迁移的影响

Effects of CENP-A on invasion and migration of ovarian cancer cells by regulating PI3K/AKT/NF-κB signaling pathway

目的探究着丝粒蛋白-A(CENP-A)对卵巢癌(OC)细胞侵袭、迁移的影响,并探讨相关机制。方法体外培养OC细胞系A2780细胞株,分为NG组(空白对照组)、pcDNA组(阴性转染组,转染pcDNA载体质粒)、pcDNA-CENP-A组(过表达CENP-A组,转染pcDNA-CENP-A载体质粒)、通路抑制剂组(转染pcDNA-CENP-A+PI3K通路抑制剂LY294002)。采用CCK-8法检测细胞增殖情况,划痕实验、Transwell实验分别检测细胞迁移、侵袭能力;免疫印迹法检测CENP-A蛋白、迁移、侵袭相关蛋白E-钙粘附蛋白(E-cadherin)、N-钙粘附蛋白(N-cadherin)及磷脂肌醇3-激酶/蛋白激酶B/核转录因子kappa B(PI3K/AKT/NF-κB)通路相关蛋白表达情况。结果A2780细胞成功转染。24 h后,随着培养时间延长,与NG组[(0.50±0.07)、(0.72±0.11)、(0.99±0.14)]、pcDNA组[(0.55±0.08)、(0.78±0.12)、(1.02±0.15)]比较,pcDNA-CENP-A组[(0.78±0.12)、(1.03±0.15)、(1.67±0.25)]、通路抑制剂组[(0.63±0.09)、(0.87±0.13)、(1.39±0.20)]A2780细胞存活力显著增加(P<0.05),与pcDNA-CENP-A组比较,通路抑制剂组A2780细胞存活力显著降低(P<0.05),呈时间依赖性。与NG组[(15.83±1.46)%、(105.32±15.78)个]、pcDNA组[(16.79±1.46)%、(108.98±16.35)个]比较,pcDNA-CENP-A组、通路抑制剂组A2780细胞迁移率[(37.96±5.80)%、(25.15±2.19)%]、侵袭数[(327.87±49.18)个、206.53±30.97)个]、CENP-A、N-cadherin、Vimentin、p-PI3K/PI3K、p-AKT/AKT、NF-κB、白细胞介素(IL-1β)、肿瘤坏死因子-α(TNF-α)蛋白表达显著增加或升高(P<0.05),E-cadherin蛋白表达显著降低(P<0.05);与pcDNA-CENP-A组比较,通路抑制剂组A2780细胞迁移率、侵袭数、CENP-A、N-cadherin、Vimentin、p-PI3K/PI3K、p-AKT/AKT、NF-κB、IL-1β、TNF-α蛋白表达显著减少或或降低(P<0.05),E-cadherin蛋白表达显著升高(P<0.05)。结论过表达CENP-A表达可促进卵巢癌细胞增殖、侵袭及迁移,可能是通过激活PI3K/AKT/NF-κB信号通路实现的。

Objective To investigate the effects of centromere protein-A(CENP-A)on the invasion and migration of ovarian cancer(OC)cells and explore the related mechanism.Methods OC cell line A2780 was cultured in vitro,and they were divided into Ng Group(Blank Control Group),pcDNA group(negative transfection group:PCDNA vector plasmid),pcDNA-CENP-A group(over-expression Group:pcDNA-CENP-A Vector Plasmid)and pathway inhibitor group(TRANSFECTION-CENP-A+PI3K pathway inhibitor LY294002).The cell proliferation was detected by CCK-8 method;the cell migration and invasion was detected by Scratch test and Transwell test;the expression of CENP-A,E-cadherin,N-cadherin and phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-kappa B(PI3K/AKT/NF-κB)pathway related proteins was detected by Western blot.Results A2780 cells were successfully transfected.After 24 hours,with the extension of culture time,compared with that in NG group[(0.50±0.07),(0.72±0.11),(0.99±0.14)]and pcDNA group[(0.55±0.08),(0.78±0.12),(1.02±0.15)],the viability of A2780 cells in pcDNA-CENP-A group[(0.78±0.12),(1.03±0.15),(1.67±0.25)]and pathway inhibitor group[(0.63±0.09),(0.87±0.13),(1.39±0.20)]increased significantly(P<0.05),compared with that in the pcDNA-CENP-A group,the viability of A2780 cells in the pathway inhibitor group was significantly decreased(P<0.05),in a time-dependent manner.Compared with those in NG group[(15.83±1.46)%,(105.32±15.78)individual]and pcDNA group[(16.79±1.46)%,(108.98±16.35)individual],the migration rate[(37.96±5.80)%,(25.15±2.19)%]and invasion number[(327.87±49.18)individual,206.53±30.97)individual]of A2780 cells,protein expression of CENP-A,N-cadherin,Vimentin,p-PI3K/PI3K,p-AKT/AKT,NF-κB,interleukin(IL-1β),tumor necrosis factor-α(TNF-α)in pcDNA-CENP-A group and pathway inhibitor group were significantly higher(P<0.05),the expression of E-cadherin was significantly lower(P<0.05);compared with those in the pcDNA-CENP-A group,the migration rate and invasion number of A2780 cells,protein expression of CENP-A,N-cadherin,Vimentin,p-PI3K/PI3K,p-AKT/AKT,NF-κB,interleukin(IL-1β),tumor necrosis factor-α(TNF-α)in pathway inhibitor group were significantly lower(P<0.05),and the expression of E-cadherin was significantly higher(P<0.05).Conclusion Overexpression of CENP-A can promote the proliferation,invasion and migration of ovarian cancer cells,which may be achieved by activating PI3K/AKT/NF-κB signaling pathway.