C1q样蛋白4通过PI3K/Akt信号通路调节乳腺癌干细胞特性和上皮间质转化

C1ql4 regulates the characteristics of breast cancer stem cells and epithelial-mesenchymal transition through PI3K/Akt signaling pathway

目的:初步探讨C1q样蛋白4(C1ql4)对乳腺癌干细胞特性及上皮间质转化的影响。方法:采用siRNA干扰技术,下调细胞株BT549中C1ql4的表达,采用RT-qPCR法及Western blot法验证C1ql4的沉默,并将后续实验分为空白组、对照组(si-NC组)及C1ql4沉默组(si-C1ql4组)。采用RT-qPCR法及Western blot法分别对各组细胞干性相关指标(CD44、Nanog、OCT4)及EMT相关指标(E-cadherin、Vimentin、N-cadherin)的mRNA和蛋白表达情况进行检测。利用流式细胞仪检测各组细胞的凋亡情况、CD44^(+)CD24^(-/low)细胞含量变化,成球试验比较各组细胞成球能力,Transwell小室实验比较各组细胞的迁移能力。Western blot法检测信号通路蛋白表达水平,探索C1ql4影响乳腺癌细胞干性及对上皮间质转化可能的机制。结果:下调C1ql4表达后,si-C1ql4组CD44、Nanog、OCT4、Vimentin及N-cadherin的mRNA和蛋白表达量,乳腺癌干细胞含量、迁移能力及成球能力均低于空白组和si-NC组;E-cadherin mRNA和蛋白表达量、细胞凋亡率均高于其他两组;PI3K和Akt的蛋白表达水平及磷酸化水平也明显低于空白组和si-NC组,差异均具有统计学意义(P<0.05),而空白组和si-NC组之间上述指标的差异无统计学意义。结论:C1ql4可促进乳腺癌细胞干性表达及乳腺癌细胞的上皮间质转化,还可增强乳腺癌细胞成球、迁移能力,抑制瘤细胞凋亡,且作用机制可能与PI3K/Akt信号通路有关。

Objective:To explore the effects of C1ql4 on the characteristics of breast cancer stem cells and the epithelial mesenchymal transition.Methods:siRNA interference technique was used to down-regulate the expression of C1ql4 in BT549 cell line.RT-qPCR and Western blot methods were used to verify the silence of C1ql4.The subsequent experiments were divided into control group,empty vector group(si-NC group)and C1ql4 silence group(si-C1ql4 group).The mRNA and protein expression of stem cell characteristic-related protein(CD44,Nanog,OCT4)and EMT-related proteins(E-cadherin,Vimentin,N-cadherin)in each group were detected,respectively.The apoptosis,CD44^(+)CD24^(-/low) cell content of cells in each group were detected by flow cytometry,and the spheroidizing ability of cells in each group was compared by spheroidizing test,and the migration ability of cells in each group was compared by Transwell chamber test.Western blot was used to detect the expression level of signal pathway proteins,and explore the possible mechanism of C1ql4 affecting the characteristics of breast cancer stem cells and epithelial mesenchymal transformation.Results:After down-regulation of C1ql4 expression,the mRNA and protein expression of CD44,Nanog,OCT4,Vimentin and N-cadherin,breast cancer stem cell ratio,balling and migration ability of si-C1ql4 group were lower than those of control group and si-NC group,while the expression of E-cadherin mRNA and protein,apoptosis rate were significantly higher than those of the other two groups.The protein expression level and phosphorylation level of PI3K and Akt were also significantly lower than those of control group and si-NC group(P<0.05),but there was no significant difference between control group and si-NC group.Conclusion:The expression of C1ql4 can promote the expression of breast cancer stem cells and the epithelial mesenchymal transformation,enhance the biological behavior of breast cancer cells such as spheroidization and migration,inhibit cell apoptosis.The mechanism of action may be related to PI3K/Akt signaling pathway.