摘要
目的探讨miR-129-1-3p对人鼻咽癌耐药细胞HNE1/顺铂(CDDP)敏感性的影响及相关作用机制。方法培养人鼻咽癌HNE1细胞及耐CDDP的HNE1/CDDP细胞,噻唑蓝(MTT)法检测HNE1/CDDP细胞对于CDDP的耐药指数;采用反转录PCR检测miR-129-1-3p和WEE1蛋白激酶在两种细胞的表达水平。采用脂质体法将miR-129-1-3p模拟物(miR-129-1-3p mimics)和阴性对照RNA(miR-NC)转染HNE1/CDDP细胞,并设立对照组。MTT法检测转染后HNE1/CDDP细胞对CDDP的半数抑制浓度(IC50)变化,Western blot法检测转染后各组细胞WEE1蛋白水平,双荧光素酶实验评价miR-129-1-3p对WEE1的靶向调控作用。结果HNE1/CDDP细胞对于CDDP的耐药指数为5.29。HNE1细胞中的miR-129-1-3p水平高于耐CDDP的HNE1/CDDP细胞,WEE1 mRNA水平低于耐CDDP的HNE1/CDDP细胞,miR-129-1-3p水平与WEE1 mRNA水平呈负相关(r=-0.9784)。与其余两组相比,miR-129-1-3p mimics转染后,HNE1/CDDP细胞对CDDP的IC50明显降低,WEE1的蛋白水平也明显降低。双荧光素酶实验结果显示miR-129-1-3p靶向调控WEE1的3'非翻译区(3'-UTR)。结论miR-129-1-3p通过靶向抑制WEE1表达逆转HNE1/CDDP鼻咽癌细胞CDDP耐药。
Objective To explore the effect of miR-129-1-3p on cisplatin sensitivity of chemo-resistant epithelial nasopharyngeal carcinoma HNE19/CDDP cells and the related mechanism.Methods Human nasopharyngeal carcinoma HNE1 cells and cisplatin-resistant HNE19/CDDP cells were cultured.Cisplatin resistance index of cisplatin-resistant HNE1/CDDP cells was tested by MTT assay.The levels of miR-129-1-3p and WEE1 mRNA in HNE1 and HNE19/CDDP cells were measured by real-time quantitative PCR.miR-129-1-3p mimics and unrelated sequence control(miR-129-1-3p NC)were transfected into HNE1/CDDP cells using LipofectamineTM 2000.The drug sensitivity(IC50)of these cells to cisplatin was determined by MTT assay.The protein expression level of WEE1 was determined by Western blot analysis.The 3'-UTR of WEE1 was cloned into luciferase reporter vector and its luciferase activity was detected to verify whether miR-129-1-3p targets WEE1.Results Resistance index of HNE1/CDDP cells to cisplatin was 5.29.The expression level of miR-129-1-3p in the HNE1 cells was significantly higher than that in the HNE1/CDDP cells.The mRNA expression level of WEE1 in the HNE1 cells was lower than that in the HNE1/CDDP cells.The level of miR-129-1-3p was negatively correlated with the level of WEE1 mRNA(r=-0.9784).The IC50 of cisplatin was significantly reduced in the HNE1/CDDP cells after transfected with miR-129-1-3p mimics.The protein expression level of WEE1 in the cells transfected with miR-129-1-3p mimics significantly decreased as compared with the control group and blank group.The miR-129-1-3p regulated the 3'-UTR of WEE1 and reduced the expression activity of luciferase.Conclusion miR-129-1-3p could reverse cisplatin resistance of HNE1/CDDP nasopharyngeal carcinoma cells via inhibiting WEE1 expression.
作者
张浩轩
陆进
蒋成义
卢伟
ZHANG Haoxuan;LU Jin;JIANG Chengyi;LU Wei(Department of Human Anatomy,Bengbu Medical College,Bengbu 233030;Anhui Provincial Key Laboratory of Tissue Transplantation,Bengbu Medical College,Bengbu 233030;First Affiliated Hospital,Bengbu Medical College,Bengbu 233030;University of Science and Technology of China,Hefei 230026,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第11期1014-1019,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
安徽省教育厅高校自然科学重点研究项目(KJ2018A0236)。
