MiR-139-5 p通过靶向CXCR4/PI3 K/Akt信号通路增强鼻咽癌顺铂耐药细胞对顺铂的敏感性

Enhancing the sensitivity of nasopharyngeal carcinoma cells to cisplatin with MiR-139-5p by targeting CXCR4/PI3K/Akt signaling pathway

目的 探讨miR-139-5p对鼻咽癌顺铂（DDP）耐药细胞DDP敏感性的影响及其相关作用机制.方法 培养鼻咽癌DDP耐药细胞株HNE1/DDP,分为miR-139-5p组、阴性对照组、DDP组和空白对照组,miR-139-5p组转染miR-139-5p类似物,阴性对照组转染阴性对照序列,DDP组和空白对照组不做转染处理.转染后miR-139-5p组、阴性对照组和DDP组均加入20μmol/L DDP,空白对照组加入培养基,溴化吡啶（PI）单染后流式细胞术检测各组HNE1/DDP细胞凋亡率.采用Western blot检测HNE1/DDP细胞miR-139-5p组、阴性对照组和空白对照组磷酸肌醇3-激酶（PI3K）、磷酸化（p）-PI3K、蛋白激酶B（Akt）、p-Akt及趋化因子受体4（CXCR4）蛋白的相对表达水平.采用实时荧光定量PCR（qPCR）检测miR-139-5p组、阴性对照组和空白对照组CXCR4 mRNA的表达水平.结果 空白对照组、DDP组、阴性对照组和miR-139-5p组HNE1/DDP细胞凋亡率分别是6.26％ ±1.19％、22.43％ ±3.88％、23.87％ ±3.21％和40.87％ ±4.04％,DDP组、阴性对照组和miR-139-5p组细胞凋亡率均高于空白对照组,miR-139-5p组细胞凋亡率高于DDP组和阴性对照组,差异均有统计学意义（P值均〈0.05）.Western blot检测显示,miR-139-5p组p-Akt、p-PI3K、PI3K及CXCR4蛋白的相对表达量均低于空白对照组和阴性对照组,差异均有统计学意义（P值均〈0.01）.实时荧光定量PCR检测结果显示,miR-139-5p组中HNE1/DDP细胞CXCR4的mRNA相对表达水平低于空白对照组和阴性对照组,差异均有统计学意义（P值均〈0.01）.结论 转染miR-139-5p可提高鼻咽癌耐药细胞株HNE1/DDP对DDP的敏感性,其作用机制可能与抑制CXCR4的表达进而调控其下游PI3K/Akt信号通路的活化有关.

Objective To investigate the effects and molecular mechanisms of miR-139-5p on cisplatin sensitivity of nasopharyngeal carcinoma cisplatin-resistant cells.Methods The cultured nasopharyngeal carcinoma cisplatin （DDP） resistant cell line HNE1/DDP cells were divided into miR-139-5p group, negative control group, DDP group and blank control group.The miR-139-5p group was transfected with miR-139-5p mimics and the negative control group was transfected with negative control sequences. The DDP group and the blank control group were not treated. After transfection, 20 μmol/L DDP was added to the miR-139-5p group, negative control group, and DDP group. The blank control group was only given the medium, and the apoptosis rate of each group of HNE1/DDP cells was detected by single staining with propiodide （PI）. The expression levels of phosphatidylinositid 3-kinase（PI3K）, p-PI3K, protein kinase B（Akt）, p-Akt and chemokine receptor 4（CXCR4） proteins in miR-139-5p group, negative control group and blank control group were detected by Western blot. The mRNA expression levels of CXCR4 in miR-139-5p group, negative control group and blank control group were detected by real-time quantitative PCR （RT-qPCR）.Results The apoptosis rate detection of HNE1/DDP cells showed that the apoptosis rate of blank control group, DDP group, negative control group and miR-139-5p group were 6.26%±1.19%, 22.43%±3.88%, 23.87%±3.21% and 40.87±4.04%, respectively. The apoptosis rates of DDP group, negative control group and miR-139-5p group were significantly higher than that of the blank control group,and the apoptosis rate of miR-139-5p group was significantly higher than those of DDP group and negative control group. The difference was statistically significant （all P values＜0.05）. Western blot analysis showed that the relative expression levels of p-Akt, p-PI3K, PI3K, and CXCR4 proteins in miR-139-5p group were significantly lower than those in blank control group and negative control group （all P values＜0.01） . The results of RT-qPCR showed that the mRNA expression level of CXCR4 in the miR-139-5p group was significantly lower than those in the blank control group and the negative control group, and the difference was statistically significant （all P values＜0.01）.Conclusions Transfection of miR-139-5p can significantly increase the sensitivity of nasopharyngeal carcinoma cell line HNE1/DDP to cisplatin, which may be related to the inhibition of the expression of CXCR4 and the regulation of the activation of its downstream PI3K/Akt signaling pathway.