鼻咽癌干细胞的生物学行为特征及CD44对Ras信号通路的调控作用

CD44 regulates biological behavior and Ras signaling pathway in nasopharyngeal carcinoma stem cells

目的探讨鼻咽癌干细胞的生物学行为和Ras信号通路特征,以及CD44与Ras信号通路活化之间的调控关系。方法采用无血清悬浮培养法获得鼻咽癌CNE2和5-8F细胞的干细胞CNE2-SC和5-8F-SC。采用细胞计数试剂盒8(CCK-8)法检测细胞增殖,细胞平板克隆形成实验观察细胞克隆形成情况,Transwell细胞迁移实验检测细胞的迁移能力,细胞黏附实验观察细胞贴壁情况,Western blot法检测细胞中Ras信号通路相关蛋白的表达水平,采用RNA干扰技术明确CD44对Ras信号通路的调控作用。结果接种后24、48和72 h,鼻咽癌干细胞CNE2-SC和5-8F-SC的增殖能力分别低于其来源鼻咽癌细胞(均P<0.05)。细胞种植14 d后,CNE2-SC细胞的克隆形成率为(44.5±1.9)%,高于其来源细胞CNE2[(34.9±1.5)%,P<0.01];5-8F-SC细胞的克隆形成率为(47.4±1.8)%,亦高于其来源细胞5-8F[(37.2±1.7)%,P<0.01]。CNE2-SC细胞的迁移细胞数为(87.6±7.8)个/视野,是CNE2细胞的3.97倍(P<0.01)。5-8F-SC细胞的迁移细胞数为(67.2±5.7)个/视野,是5-8F细胞的3.07倍(P<0.01)。接种后3 h,CNE2-SC和CNE2细胞的黏附贴壁率分别为(42.1±7.6)%和(8.9±2.0)%;接种后6 h,分别为(82.4±5.0)%和(12.1±2.2)%。CNE2-SC细胞的黏附贴壁率均高于CNE2细胞(均P<0.01)。接种后3 h,5-8F-SC和5-8F细胞的黏附贴壁率分别为(53.6±6.1)%和(7.3±1.5)%;接种后6 h,分别为(90.7±3.6)%和(11.0±1.2)%。5-8F-SC细胞的黏附贴壁率均高于5-8F细胞(均P<0.01)。与普通鼻咽癌细胞相比,鼻咽癌干细胞中CD44、Ras和N-钙黏蛋白(N-cadherin)的表达水平升高(均P<0.01),E-钙黏蛋白(E-cadherin)、10号染色体缺失的磷酸酶和张力蛋白同源物(PTEN)表达水平降低(均P<0.01),磷酸化丝裂原细胞外激酶1/2(p-MEK1/2)、磷酸化细胞外信号调节蛋白激酶1/2(p-ERK1/2)磷酸化水平升高(均P<0.01)。相关分析显示,CNE2细胞与CNE2-SC细胞中,5-8F细胞与5-8F-SC细胞中,CD44与Ras蛋白表达水平均呈高度正相关(r=0.985,P=0.002;r=0.962,P=0.038)。沉默CNE2-SC细胞中CD44的表达后,人表皮生长因子受体2(HER-2)和Ras的蛋白表达水平显著降低,p-ERK1/2、p-Akt和p-PKCδ磷酸化水平下降(均P<0.01)。结论相对于来源鼻咽癌细胞,鼻咽癌干细胞增殖能力降低,但其克隆形成能力、迁移能力、黏附能力增强,可能与其CD44调控的Ras信号通路异常激活有关。

Objective To study the biological behavior of nasopharyngeal carcinoma stem cells and to explore the activation of Ras signaling pathway regulated by CD44.Methods CNE2-SC and 5-8F-SC were nasopharyngeal carcinoma stem cells and obtained by serum-free suspension culture.Cell counting kit-8(CCK-8)assay,colony formation assay,Transwell migration assay,cell adhesion array were used to investigate the growth,proliferation,migration and adhesion of nasopharyngeal carcinoma stem cells.Western blot test was used to detect the expressions of Ras signaling pathway related proteins and siRNA-mediated interference was used to determine the activation of Ras signaling pathway regulated by CD44.Results The growth rates of CNE2-SC and 5-8F-SC cells were significantly lower than those of nasopharyngeal carcinoma cells at 24,48 and 72 hours after inoculation(P<0.05).After 14 days of implantation,the colony formation rates of CNE2-SC(44.5±1.9)%and 5-8F-SC(47.4±1.8)%were higher than those of CNE2(34.9±1.5)%and 5-8F(37.2±1.7)%,respectively(P<0.01).The migration cell number of CNE2-SC was(87.6±7.8),3.97 times higher than that of CNE2(P<0.01).The migration cell number of 5-8F-SC was(67.2±5.7),3.07 times higher than 5-8F(P<0.01).The adhesion rates of CNE2-SC and CNE2 cells were(42.1±7.6)%and(8.9±2.0)%,respectively at 3 hours after inoculation and were(82.4±5.0)%and(12.1±2.2)%at 6 hours after inoculation,respectively.The adhesion rate of CNE2-SC cells was higher than that of CNE2 cells(all P<0.01).The adhesion rates of 5-8F-SC and 5-8F cells were(53.6±6.1)%and(7.3±1.5)%at 3 hours after inoculation,and(90.7±3.6)%and(11.0±1.2)%at 6 hours after inoculation,respectively.The adhesion rate of 5-8F-SC cells was higher than that of 5-8F cells(P<0.01).The expression levels of CD44,Ras and N-cadherin were significantly higher,while phosphatase and tensin homolog deleted on chromosome 10(PTEN),E-cadherin in nasopharyngeal carcinoma stem cells were lower than those of the nasopharyngeal carcinoma cells.Furthermore,the levels of phosphorylated mitogen extracellular kinase1/2(p-MEK1/2)and phosphorylated extracellular signal-regulated protein kinase1/2(p-ERK1/2)were significantly increased in nasopharyngeal carcinoma stem cells(P<0.01).Correlation analysis showed that the protein expression levels of CD44 was highly positively correlated with RAS in nasopharyngeal carcinoma stem cells(r=0.985,P=0.002;r=0.962,P=0.038).Deletion of CD44 in CNE2-SC decreased the expression levels of HER-2,Ras and p-ERK1/2,p-Akt and phosphorylated protein kinase C-δ(p-PKCδ)(P<0.01).Conclusions Despite compare to the nasopharyngeal carcinoma cell,nasopharyngeal carcinoma stem cells grows at a relatively slow rate,the capacities of clone formation,migration,adhesion are promoted.This may be related to the CD44-regulated abnormal activation of Ras signaling pathway.