β-catenin过表达对胰腺癌干细胞生物学特性的影响

Effects ofβ-catenin overexpression on biological characteristics of pancreatic cancer stem cells

目的分析β-catenin蛋白在胰腺癌干细胞恶性生物学特性维持过程中的作用及其机制。方法分离培养原代胰腺癌细胞,将胰腺癌细胞分为NC组和ADβ-catenin组,NC组细胞转染阴性空载病毒,ADβ-catenin组细胞转染β-catenin过表达慢病毒载体。采用流式细胞仪分析细胞株肿瘤干细胞表面标志物CD24、CD44及ESA的表达情况;分选出CD24^(+)CD44^(+)ESA^(+)细胞并扩大培养,通过检测分选出细胞的成球能力、克隆形成能力、增殖能力、侵袭迁移能力、细胞周期及凋亡情况;分析β-catenin过表达对胰腺癌干细胞生物学特性的影响。结果ADβ-catenin组细胞中β-catenin mRNA和蛋白的相对表达量显著高于NC组(P=0.016,P=0.015)。流式细胞仪分析结果显示,ADβ-catenin组细胞中CD24^(+)及CD24^(+)CD44^(+)ESA^(+)的细胞率显著高于NC组(P<0.05)。与NC组比较,ADβ-catenin组胰腺癌干细胞克隆形成能力、成球能力、侵袭与迁移细胞数均显著增加(P<0.05)。ADβ-catenin组细胞在24 h、48 h及72 h的光密度(OD)值均显著高于NC组(P<0.05)。与NC组相比,ADβ-catenin组G_(0)/G_(1)期细胞比例显著减少(P<0.05),S期细胞比例增高(P<0.05)。ADβ-catenin组的细胞凋亡率显著低于NC组(P<0.05)。结论β-catenin通过提高胰腺癌原代细胞中CD24^(+)CD44^(+)ESA^(+)的细胞率,促进胰腺癌干细胞成球能力,增强细胞增殖、侵袭与迁移能力,抑制细胞的凋亡,从而维持胰腺癌干细胞的恶性生物学特性。

Objective To investigate the role and mechanism ofβ-catenin protein in the maintenance of malignant biological characteristics of pancreatic cancer stem cells.Methods Primary pancreatic cancer cells were isolated and cultured,then divided into the negative control(NC)group and the ADβ-catenin group,cells in the NC group were transfected with negative empty vectors,and cells in the ADβ-catenin group were transfected withβ-catenin overexpression lentiviral vectors.Flow cytometry was used to analyze the expression of tumor stem cell surface markers CD24,CD44,and ESA.CD24^(+)CD44^(+)ESA^(+)cells were sorted and expanded in culture,and their sphereforming ability,colony-forming ability,proliferation ability,invasion ability and migration ability,cell cycle,and apoptosis were assessed.The impact ofβ-catenin overexpression on the biological characteristics of pancreatic cancer stem cells was analyzed.Results The relative expression levels ofβ-catenin mRNA and protein in the ADβ-catenin group were significantly higher than those in the NC group(P=0.016,P=0.015).Flow cytometry analysis revealed that the percentage of CD24^(+)and CD24^(+)CD44^(+)ESA^(+)cells in the ADβ-catenin group was significantly higher than that in the NC group(P<0.05).Compared with the NC group,the colony-forming ability,sphere-forming ability,numbers of invasion and migration of pancreatic cancer stem cells in the ADβ-catenin group were significantly increased(P<0.05).The optical density(OD)values at 24 hours,48 hours,and 72 hours were significantly higher in the ADβ-catenin group than those in the NC group(P<0.05).Compared with the NC group,the proportion of G_(0)/G_(1) phase cells in the ADβ-catenin group was significantly decreased(P<0.05),and the proportion of S phase cells was increased(P<0.05).The apoptosis rate in the ADβ-catenin group was significantly lower than that in the NC group(P<0.05).Conclusionβ-catenin can improve the percentage of CD24^(+)CD44^(+)ESA^(+)cells in primary pancreatic cancer cells,promote sphere-forming ability,enhance cell proliferation,invasion,and migration,and inhibit cell apoptosis,thereby maintaining the malignant biological characteristics of pancreatic cancer stem cells.