摘要
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.
作者
邵倩倩
马莹晔
李慧
王晓敏
张明洁
陈德尚
陆兆屹
孟洁
马士崟
Shao Qiarutian;Ma Yingye;Li Hui;Wang Xiaoming;Zhartg Mingjie;Chert Deshang;Lu Zhaoyi;Mertg Jie;Ma Shiyin(Graduate School, Bertgbu Medical College, Bertgbu 233030, China)
出处
《中华解剖与临床杂志》
2018年第2期149-154,共6页
Chinese Journal of Anatomy and Clinics
基金
安徽省自然科学基金(1608085MH236)
关键词
鼻咽肿瘤
抗药性
肿瘤
微小RNA
顺铂
趋化因子受体4
磷酸肌醇3-激酶
蛋白激酶B
Nasopharyngeal neoplasms
Drug resistance
neoplasm
Micro RNA
Cisplatin
Phosphatidylinositid 3-kinase
Protein kinase B
Chemokine receptor 4