神经纤毛以及类似透拉蛋白2对胆囊癌细胞增殖、迁移、细胞周期和凋亡的影响及相关机制研究

Effects of neuropilin and tolloid-like 2 on proliferation, migration, cell cycle and apoptosis of gallbladder carcinoma cells and related mechanism

目的探讨神经纤毛以及类似透拉蛋白2(NETO2)对胆囊癌细胞增殖、迁移、周期和凋亡的影响及分子机制。方法通过实时荧光定量聚合酶链反应(qPCR)和免疫印迹法分别检测胆囊癌细胞系GBC-SD、ZJU-0430、NOZ中NETO2的mRNA及蛋白表达水平,利用质粒转染法构建NETO2过表达及敲减的稳转细胞株。通过细胞计数法(CCK-8)、克隆形成实验、细胞迁移实验、流式细胞术和免疫印迹法检测NETO2对胆囊癌细胞增殖、克隆形成能力、迁移、周期进程、凋亡和上皮间质转化(EMT)的影响,以及磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路的变化。结果成功构建NETO2基因过表达的胆囊癌GBC-SD及ZJU-0430细胞,NETO2基因敲减的NOZ细胞。在ZJU-0430、GBC-SD细胞过表达NETO2后,胆囊癌细胞的增殖及迁移能力提高;可促进细胞从G0/G1期向S期进展,并抑制细胞凋亡;细胞克隆数分别由(78.5±9.2)、(217.0±6.4)增加为(213.5±10.3)、(296.3±9.3)(t=10.98、6.51,P=0.008、0.023);迁移细胞数分别由(100倍视野)(198.6±8.4)个、(233.3±11.0)个增加为(382.7±12.4)个、(379.0±7.3)个(t=16.98、16.85,均P<0.001);细胞总凋亡率由(29.7±0.9)%、(35.6±1.1)%降低为(19.2±0.5)%、(29.1±0.4)%(t=9.74、9.05,均P<0.001);EMT相关蛋白如N-钙粘蛋白、波形蛋白、锌指转录因子Snail、Slug表达上调,而E-钙粘蛋白表达下调;PI3K/Akt通路磷酸化蛋白p-PI3K及p-Akt表达上调,上述变化差异均具有统计学意义(均P<0.05)。而敲减NETO2表达则导致相反的变化。结论NETO2通过调控PI3K/Akt信号通路影响EMT进程,从而促进胆囊癌细胞的增殖和迁移,促进癌细胞周期进程并抑制癌细胞凋亡。

Objective To investigate the effects and molecular mechanism of neuropilin and tolloid-like 2(NETO2)on proliferation,migration,cell cycle,and apoptosis in gallbladder cancer(GBC).Methods The NETO2 mRNA and protein expression in GBC-SD,ZJU-0430,NOZ GBC cells were detected by quantitative real-time polymerase chain reaction and Western blot.NETO2 overexpression and knockdown stable cell lines were constructed by plasmid transfection.Cell counting kit-8 assay,colony formation assay,transwell assay,flow cytometry and WB assay were performed to evaluate proliferation,migration,cell cycle,apoptosis,epithelial-mesenchymal transition(EMT)and changes of phosphatidylinositol-3 kinase/protein kinase B(PI3K/Akt)signaling pathway.Results GBC-SD and ZJU-0430 cells with NETO2 gene overexpression and NOZ cells with NETO2 gene knockdown were effectively constructed.NETO2 overexpression in gallbladder cancer cell lines significantly improved cell proliferation and migration,advanced cell cycle progression from G0/G1 to S phase,and inhibited cell apoptosis.In the ZJU-0430 and GBC-SD cells,the clone number increased from(78.5±9.2),(217.0±6.4)to(213.5±10.3),(296.3±9.3)(t=10.98,6.51;P=0.008,0.023);The number of migrating cells increased from(198.6±8.4),(233.3±11.0)to(382.7±12.4),(379.0±7.3)(t=16.98,16.85,both P<0.001);The total apoptosis rate reduced from(29.7±0.9)%,(35.6±1.1)%to(19.2±0.5)%,(29.1±0.4)%(t=9.74,9.05;both P<0.001);The expression of EMT related proteins such as N-cadherin,Vimentin,Snail,and Slug were upregulated,while E-cadherin expression was downregulated.Phosphorylated PI3K(p-PI3K)and Akt(p-Akt)protein expression were significantly increased(all P<0.05).In contrast,NETO2 knockdown had the opposite effect on all these parameters.Conclusion NETO2 influences the EMT process by regulating the PI3K/Akt signaling pathway,thus promotes GBC cell proliferation,migration and cell cycle progression,and inhibits cancer cell apoptosis.