LEF1介导MDR1/P-gp调控人结肠癌细胞多药耐药的研究  

LEF1 regulates MDR1/P-gp-mediated multidrug resistance in colon cancer cells

作　　者：王文娟[1] 刘梦洁[1] 徐瑞[2] 王淑红[1] 南克俊[1] WANG Wenjuan;LIU Mengjie;XU Rui;WANG Shuhong;NAN Kejun(Department of Oncology,First Affiliated Hospital of Xi＇an Jiaotong University,Xi＇an 710061,China;Department of Internal Medicine,Affiliated Hospital of Medical College of Xi＇an Jiaotong University,Shaanxi Province Cancer Hospital)

机构地区：[1]西安交通大学第一附属医院肿瘤内科,西安710061 [2]西安交通大学医学院附属陕西省肿瘤医院内一科

出　　处：《山西医科大学学报》2018年第8期905-911,共7页Journal of Shanxi Medical University

基　　金：国家自然科学基金资助项目(81502099);中央高校基本科研业务费专项基金资助项目(xjj2015027)

摘　　要：目的通过调节人结肠癌细胞HCT116及HCT116/L多药耐药细胞中LEF1(lymphoid enhancer factor-1,LEF1)的表达,以探讨LEF1对人结肠癌细胞多药耐药(multidrug resistance,MDR)的影响及其对P-糖蛋白(P-glycoprotein,P-gp)的调控作用。方法应用LEF1过表达质粒pc DNA-LEF1和LEF1 siRNA上调及沉默人结肠癌细胞HCT116和奥沙利铂耐药细胞HCT116/L中的LEF1的表达,MTT法检测LEF1对HCT116和HCT116/L细胞化疗药物敏感性的影响;应用MDR1过表达质粒pc DNA-MDR1和MDR1 siRNA上调及沉默人结肠癌细胞HCT116-LEF1细胞(过表达LEF1细胞株)和HCT116/L-si LEF1(沉默LEF1奥沙利铂耐药细胞株)的MDR1的表达,MTT法检测MDR1对HCT116-LEF1细胞和HCT116/L-si LEF1细胞化疗药物敏感性的影响;real time-PCR、Western blot等方法检测多药耐药基因MDR1和P-gp蛋白的表达。结果 HCT116/L细胞中奥沙利铂(oxaliplatin,L-OHP)和5-氟尿嘧啶(5-fluorouracil,5-FU)的半数抑制浓度(IC_(50))高于HCT116细胞(P<0.05),其LEF1、MDR1 mRNA和P-gp蛋白的表达水平均高于HCT116细胞(P<0.05)。过表达LEF1后,HCT116细胞对奥沙利铂和5-氟尿嘧啶的IC_(50)分别由(15.04±2.68)μmol/L和(5.89±2.77)μmol/L上升至(74.16±9.05)μmol/L和(35.02±6.19)μmol/L(P<0.05),P-pg的蛋白表达水平亦明显升高(P<0.05);沉默LEF1表达后,HCT116/L细胞对奥沙利铂和5-氟尿嘧啶的IC_(50)分别由(511.89±37.79)μmol/L和(220.22±66.33)μmol/L下降至(253.56±71.42)μmol/L和(94.94±25.45)μmol/L(P<0.05),P-gp的蛋白表达水平明显降低(P<0.05)。沉默MDR1表达后,HCT116-LEF1细胞对奥沙利铂和5-氟尿嘧啶的IC_(50)分别由(85.68±8.46)μmol/L和(36.92±3.02)μmol/L下降至(29.86±5.93)μmol/L和(7.61±1.47)μmol/L(P<0.05)。过表达MDR1后,HCT116/L-si LEF1细胞对奥沙利铂和5-氟尿嘧啶的IC_(50)分别由(216.55±49.63)μmol/L和(101.48±17.93)μmol/L上升至(351.46±23.31)μmol/L和(175.23±28.25)μmol/L(P<0.05)。结论 LEF1通过介导MDR1/P-gp的表达调控人结肠癌细胞HCT116对奥沙利Objective To explore the effects of LEF1 expression on multidrug resistance and expression of P-gp in colon cancer cells HCT116 and HCT116/L.Methods LEF1-overexpressed plasmid pc DNA-LEF1 and LEF1 siRNA were used to upregulate and silence the expression of LEF1 in HCT116 and HCT116/L cells,respectively.MDR1-overexpressed plasmid pc DNA-MDR1 and MDR1 siRNA were used to upregulate and silence the expression of MDR1 in HCT116/L-si LEF1 and HCT116-LEF1 cells,respectively.The sensitivities of MDR1 to oxaliplatin and 5-flurouacil were evaluated by MTT assay.MDR1 mRNA and P-gp expression were evaluated by real-time fluorescence quantitative PCR（RT-PCR） and Western blot.Results IC50 of oxaliplatin and 5-flurouacil in HCT116/L cells were higher than that in HCT116 cells（P〈 0.05）.The expression of LEF1,MDR1 mRNA and P-gp in HCT116/L cells was higher than that in HCT116 cells（P〈 0.05）.After over-expressing LEF1,IC50 of oxaliplatin and 5-flurouacil in LEF1-overexpressed HCT116 cells increased from（15.04 ±2.68） μmol/L and（5.89 ±2.77） μmol/L to（74.16 ±9.05） μmol/L and（35.02 ±6.19） μmol/L,respectively（P 〈0.05）,and the expression of P-gp in HCT116-LEF1 cell was also up-regulated（P〈 0.05）.After silencing the expression of LEF1 in HCT116/L cell,IC50 of oxaliplatin and 5-flurouacil in LEF1-silenced HCT116/L cells decreased from（511.89 ±37.79） μmol/L and（220.22±66.33） μmol/L to（253.56 ±71.42） μmol/L and（94.94 ± 25.45） μmol/L（P〈 0.05）,respectively,and the expression of P-gp in HCT116/L-si LEF1 cell was down-regulated（P 0.05）.After silencing the expression of MDR1,IC50 of oxaliplatin and 5-flurouacil in MDR1-silenced HCT116-LEF1 cells decreased from（85.68±8.46） μmol/L and（36.92±3.02） μmol/L to（29.86±5.93） μmol/L and（7.61±1.47） μmol/L（P〈 0.05）,respectively.After over-expressing MDR1,IC50 of oxaliplatin and 5-flurouacil in MDR1-overexpressed HCT116/L-si LEF1 cells increased from（216.55±49.63） μmol/L and（101.48±17.

关 键 词：LEF1 MDR1/P-GP 结肠癌 多药耐药 

分 类 号：R735.35[医药卫生—肿瘤]