芬太尼通过Wnt3a/β-连环蛋白信号通路促进乳腺癌细胞干细胞特性的研究

Study on fentanyl effect in promoting the stemness of breast cancer cell via Wnt3a/β-catenin signaling pathway

目的研究芬太尼对乳腺癌细胞干细胞特性(简称干性)的影响及分子机制。方法2018年1月至2019年10月采用人乳腺癌细胞系BT549作为体外研究对象,经0.01和0.10μmol/L芬太尼处理后,通过成球能力实验和克隆形成能力实验检测乳腺癌细胞干性的变化;采用实时定量聚合酶链反应(FQ-PCR)检测干性相关转录因子性别决定区Y框蛋白2(Sox2)、八聚体结合转录因子4(Oct4)和Nanog mRNA表达水平;利用蛋白免疫印迹实验检测Wnt信号通路关键蛋白Wnt3a、磷酸化糖原合酶激酶-3β(p-GSK-3β)、糖原合酶激酶-3β(GSK-3β)、β-连环蛋白(β-catenin)的表达水平。下调Wnt3a后,再行蛋白免疫印迹实验和成球能力实验。结果0.01和0.10μmol/L芬太尼处理的乳腺癌细胞球体直径、克隆形成率、Sox2 mRNA、Oct4 mRNA、Nanog mRNA、Wnt3a、p-GSK-3β、GSK-3β和β-catenin明显大于空白对照[(131.22±1.06)和(636.37±0.02)μm比(72.68±0.13)μm、(41.33±0.03)%和(60.58±1.08)%比(20.93±0.15)%、2.25±0.20和3.82±0.84比1.00、1.87±1.06和3.35±0.04比1.00、2.85±0.03和4.36±0.50比1.00、1.82±0.03和2.57±0.42比1.00、2.04±0.13和2.81±0.05比1.00、1.62±0.17和2.93±0.06比1.00、2.15±0.02和3.54±0.21比1.00],0.10μmol/L芬太尼处理的乳腺癌细胞各指标明显大于0.01μmol/L芬太尼处理的乳腺癌细胞,差异有统计学意义(P<0.01)。干扰Wnt3a后,p-GSK-3β、GSK-3β、β-catenin、Sox2、Oct4和球体直径的表达明显低于空白对照[0.12±0.05比1.00、0.53±0.06比1.00和0.24±0.21比1.00、0.28±0.10比1.00和0.06±0.01比1.00、(18.14±0.30)μm比(74.32±0.12)μm],差异有统计学意义(P<0.01)。结论芬太尼通过激活Wnt3a/β-catenin信号通路促进乳腺癌细胞干性。

Objective To explore the effect and mechanism of fentanyl on promoting the stemness of breast cancer cell.Methods From January 2018 to October 2019,human breast cancer cell line BT549 was used as the in vitro research object.Breast cancer cell BT549 was pretreated with 0.01 and 0.10μmol/L fentanyl.Sphere formation assay and colony formation assay were performed to investigate the role of fentanyl on breast cancer cell stemness.Fluorescent quantitative polymerase chain reaction(FQ-PCR)was used to detect the mRNA level of stemness-related transcription factors gender-determining area Y box protein 2(Sox2),octamer binding transcription factor 4(Oct4)and Nanog.Western blotting assay was performed to determine the level of Wnt3a/β-catenin pathway-related markers Wnt3a,phosphorylated glycogen synthase kinase-3β(p-GSK-3β),glycogenase kinase-3β(GSK-3β)andβ-catenin.After down-regulating Wnt3a,western blotting assay and sphere formation assay were performed.Results The sphere diameter,colony formation rate and the expression of Sox2 mRNA,Oct4 mRNA,Nanog mRNA,Wnt3a,p-GSK-3β,GSK-3βandβ-catenin in 0.01 and 0.10μmol/L fentanyl-treated breast cancer cell were significant higher than those in blank control:(131.22±1.06)and(636.37±0.02)μm vs.(72.68±0.13)μm,(41.33±0.03)%and(60.58±1.08)%vs.(20.93±0.15)%,2.25±0.20 and 3.82±0.84 vs.1.00,1.87±1.06 and 3.35±0.04 vs.1.00,2.85±0.03 and 4.36±0.50 vs.1.00,1.82±0.03 and 2.57±0.42 vs.1.00,2.04±0.13 and 2.81±0.05 vs.1.00,1.62±0.17 and 2.93±0.06 vs.1.00,2.15±0.02 and 3.54±0.21 vs.1.00,the indexes in 0.10μmol/L fentanyl-treated breast cancer cell were significantly higher than those in 0.01μmol/L fentanyl-treated breast cancer cell,and there were statistical differences(P<0.01).After down-regulating Wnt3a,the expressions of p-GSK-3β,GSK-3β,β-catenin,Sox2,Oct4 and sphere diameter were significantly lower than those in blank control:0.12±0.05 vs.1.00,0.53±0.06 vs.1.00 and 0.24±0.21 vs.1.00,0.28±0.10 vs.1.00 and 0.06±0.01 vs.1.00,(18.14±0.30)μm vs.(74.32±0.12)μm,and there were statistical differences(P<0.01).Conclusions Fentanyl promotes breast cancer cell stemness by Wnt3a/β-catenin signaling pathway.