抑制miR-130a表达对SACC-83顺铂化疗敏感性及XIAP MDR1蛋白表达的影响
Effect of inhibiting miR-130a expression on chemosensitivity of cisplatin of SACC-83 cells and protein expressions of XIAP and MDR1
石佳佩 1王秋林 1易笑冉 1刘俊杰 1严梦 1李鹏程2
作者信息
- 1. 华中科技大学同济医学院附属协和医院口腔颌面外科,湖北武汉 430022
- 2. 华中科技大学同济医学院附属协和医院肿瘤中心,湖北武汉 430022
- 折叠
摘要
目的 研究抑制miR-130a表达对人涎腺腺样囊性癌细胞株(SACC-83)顺铂(DDP)化疗敏感性及X染色体连锁凋亡抑制蛋白(XIAP)、多药耐药基因(MDR1)表达的影响.方法 构建人涎腺腺样囊性癌DDP耐药细胞株(SACC-83/DDP),采用实时荧光定量(qRT-PCR)检测SACC-83、SACC-83/DDP细胞中miR-130a表达水平;将 SACC-83/DDP 细胞随机分为对照组、mir-130a inhibitor 组(转染 mir-130a inhibitor)、DDP+mir-130a inhibi-tor 阴性对照组(30 µmol/L DDP+转染 mir-130a inhibitor 阴性对照)及 DDP+mir-130a inhibitor 组(30 μmol/L DDP+转染mir-130a inhibitor),采用CCK-8法测定各组细胞生存率以及对DDP的耐药性,流式细胞术检测细胞凋亡率,TUNEL染色检测细胞凋亡指数;qRT-PCR检测细胞miR-130a、XIAP、MDR1、PTEN mRNA水平,免疫印记法检测细胞XIAP、MDR1、PTEN蛋白表达.结果 与SACC-83细胞相比,miR-130a在SACC-83/DDP细胞中高表达(P＜0.05);与对照组和mir-130a inhibitor组相比,DDP+mir-130a inhibitor组细胞生存率降低,凋亡率和凋亡指数升高(P＜0.05);与mir-130a inhibitor组相比,DDP+mir-130a inhibitor组细胞生存率降低,凋亡率和凋亡指数升高(P＜0.05);与对照组相比,mir-130a inhibitor 组、DDP+mir-130a inhibitor 组细胞 miR-130a 水平、XIAP mRNA及蛋白、MDR1 mRNA及蛋白表达降低(P＜0.05),凋亡率、凋亡指数和PTEN蛋白表达升高(P＜0.05);与 DPP+mir-130a inhibitor 组相比,DDP+mir-130a inhibitor 阴性对照组细胞 miR-130a 水平、XIAP mRNA 及蛋白、MDR1 mRNA及蛋白表达升高(P＜0.05),凋亡率、凋亡指数和PTEN蛋白表达下降(P＜0.05);SACC-83/DDP细胞对DDP的耐药性明显高于SACC-83细胞,抑制miR-130a表达逆转了 SACC-83/DDP细胞的DDP耐药性.结论 抑制miR-130a表达,可能下调XIAP表达,增强人涎腺腺样囊性癌细胞株对顺铂化疗的敏感性.
Abstract
Objective To investigate the effects of inhibiting miR-130a expression on the chemosensitivity of cisplatin and the expressions of X-linked inhibitor of apoptosis protein(XIAP)and multi-drug resistance(MDR1)in human salivary adenoid cystic carcinoma cell line(SACC-83).Methods Cisplatin-resistant(SACC-83/DDP)cell line was established.Real-time fluorescence quantitative analysis(qRT-PCR)was used to detect miR-130a expression level in SACC-83 and SACC-83/DDP cells.SACC-83/DDP cells were randomly divided into control group,miR-130a inhibitor group(transfected with miR-130a inhibitor),cisplatin+miR-130a inhibitor negative control group(30 μmol/L cisplatin+transfected with miR-130a inhibitor negative control)and cisplatin+miR-130a inhibitor group(30 μmol/L cisplatin+transfected with miR-130a inhibitor).CCK-8 assay was used to measure cell survival rate and cisplatin resistance.Flow cytometry was applied to measure cellular apoptosis rate.TUNEL staining was used to examine cellular apoptosis index.qRT-PCR was performed to detect mRNA levels of miR-130a,XIAP,MDR1,and PTEN.Immunoblotting was used to detect protein expressions of XIAP,MDR1 and PTEN.Results When compared to SACC-83 cells,miR-130a was highly expressed in SACC-83/DDP cells(P＜0.05).When compared to control group,cell survival rate of cisplatin+mir-130a inhibitor group was decreased,while the apoptosis rate and apoptosis index were increased(P＜0.05).When compared to miR-130a inhibitor group,cell survival rate of cisplatin+miR-130a inhibitor group was decreased,while the apoptosis rate and apoptosis index were increased(P＜0.05).When compared to control group,miR-130a levels,XIAP mRNA and protein,MDR1 mRNA and protein were decreased in miR-130a inhibitor group and cisplatin+miR-130a inhibitor group(P＜0.05),while PTEN protein expression was increased(P＜0.05).When compared to miR-130a inhibitor group,miR-130a level,XIAP mRNA and protein,MDR1 mRNA and protein expression were increased in cisplatin+miR-130a inhibitor negative control group(P＜0.05),while PTEN protein was decreased(P＜0.05).The cisplatin resistance of SACC-83/DDP cells was significantly higher than that of SACC-83 cells.Inhibiting miR-130a expression reversed cisplatin resistance of SACC-83/DDP cells.Conclusions Inhibiting miR-130 a expression may downregulate XIAP expression and enhance the sensitivity of human salivary gland adenoid cystic carcinoma cell line to cisplatin chemotherapy.
关键词
miR-130a/X染色体连锁凋亡抑制蛋白/人涎腺腺样囊性癌细胞株/顺铂/化疗敏感性Key words
miR-130a/X-linked inhibitor of apoptosis protein/human salivary adenoid cystic carcinoma cell line/cisplatin/chemosensitivity引用本文复制引用
基金项目
湖北省卫生健康委科研项目(WJ2021M121)
出版年
2024
NETL
NSTL
万方数据