[目的]探讨宫颈癌紫杉醇抗性形成的潜在机制.[方法]紫杉醇浓度从0.001 μmol/L逐渐递增至0.1 µmol/L处理Hela细胞超过12个月进行Hela紫杉醇抗性细胞(Hela/TR)的筛选.检测Hela与Hela/TR细胞的细胞活力、凋亡水平、细胞周期和肿瘤干细胞水平.紫杉醇处理后,检测Hela与Hela/TR细胞内紫杉醇浓度.[结果]紫杉醇处理可以显著抑制Hela细胞的增殖并且诱导细胞凋亡.与Hela细胞相比,Hela/TR细胞中CD44+细胞的比例显著增加,并且Hela/TR细胞中肿瘤干细胞标志物SOX2和ALDH1的表达水平高于Hela细胞.与Hela细胞相比,Hela/TR细胞的细胞内紫杉醇浓度明显降低(0.0759±0.0130 vs 0.0031±0.0004,t=12.52,P＜0.05).与 Hela 细胞相比,Hela/TR细胞中 ABCC5 的表达水平显著上升(0.15±0.04 vs 0.72±0.04,t=22.53,P＜0.05).敲低 ABCC5 后,Hela/TR 细胞的细胞内紫杉醇浓度明显升高.与Hela细胞相比,Hela/TR细胞中FOXM1的表达水平上升.敲低FOXM1后,Hela/TR细胞中ABCC5的表达水平下降(0.13±0.07 vs 0.64±0.03,4=14.97,P＜0.05),细胞内紫杉醇浓度明显上升.过表达miR-548o-3p后,Hela/TR细胞中FOXM1和ABCC5的水平表达下降,细胞内紫杉醇浓度明显上升(0.003±0.000 vs 0.072±0.010,t=15.43,P＜0.05).miR-548o-3p 靶向 FOXM1 mRNA 的 3'端非翻译区.过表达 miR-548o-3p后,Hela/TR细胞对紫杉醇的抵抗显著下降(0.51±0.05 vs 0.10±0.01,t=17.98,P＜0.05).[结论]宫颈癌紫杉醇抗性细胞中miR-548o-3p负反馈失调导致FOXM1/ABCC5轴过度激活,提升了紫杉醇从宫颈癌细胞内的排除效率,增强了宫颈癌细胞的活力.
Mechanism of miR-548o-3p negative feedback imbalance leading to paclitaxel resistance in cervical cancer
[Objective]To explore the potential mechanism of paclitaxel resistance in cervical cancer.[Method]The paclitaxel concentration was increased from 0.001 μmol/L to 0.1 μmol/L to treat Hela cells for over 12 months for the selection of Hela-paclitaxel-resistant cells(Hela/TR).The cell viability,apoptosis level,cell cycle and tumor stem cell level of Hela and Hela/TR cells were detected.After paclitaxel treatment,the intracellular paclitaxel concentration in Hela and Hela/TR cells was detected.[Result]Paclitaxel treatment significantly inhibited the proliferation and induced apoptosis of Hela cells.The proportion of CD44+cells was significantly increased in Hela/TR cells compared with Hela cells,and the expression levels of cancer stem cell markers SOX2 and ALDH1 were much higher in Hela/TR cells than in Hela cells.The intracellular paclitaxel concentration of Hela/TR cells was significantly lower than that of Hela cells(0.0759±0.0130 vs 0.0031±0.0004,t=12.52,P＜0.05).Compared with Hela cells,the expression level of ABCC5 in Hela/TR cells was significantly increased(0.15±0.04 vs 0.72±0.04,t=22.53,P＜0.05).After knockdown of ABCC5,the intracellular concentration of paclitaxel in Hela/TR cells was significantly increased.The expression level of FOXM1 was increased in Hela/TR cells compared with Hela cells.After knockdown of FOXM1,the expression level of ABCC5 in Hela/TR cells decreased(0.13±0.07 vs 0.64±0.03,t=14.97,P＜0.05),and the intracellular concentration of paclitaxel increased significantly.After over-expression of miR-548o-3p,the expression levels of FOXM1 and ABCC5 in Hela/TR cells were decreased,and the intracellular concentration of paclitaxel was significantly increased(0.003±0.000 vs 0.072±0.010,t=15.43,P＜0.05).miR-548o-3p targeted the 3'untranslated region of FOXM1 mRNA.After overexpression of miR-548o-3p,the resistance of Hela/TR cells to paclitaxel was significant-ly decreased(0.51±0.05 vs 0.10±0.01,t=17.98,P＜0.05).[Conclusion]The negative feedback dysregulation of miR-548o-3p in cervical cancer paclitaxel-resistant cells leads to excessive activation of FOXM1/ABCC5 axis,which improves the elimination efficiency of paclitaxel from cervical cancer cells and enhances the viability of cervical cancer cells.
NETL
NSTL
万方数据
