CAJ | 학술논문

目的检测小干扰RNA(siRNA)对肝癌耐药细胞株HepG2/阿霉素(ADM)多药耐药相关蛋白基因2(MRP2)及其蛋白产物的抑制作用,观察对多药耐药性的影响.方法使用浓度梯度法制作HepG2耐ADM细胞株(HepG2/ADM),ADM浓度从0.1～2.0 m/L:设计合成针对MRP2的siRNA小片段,转染HepG2/ADM耐药细胞,24 h后通过实时荧光定量逆转录-聚合酶链反应(RT-qPCR)和Western blot检测siRNA对MRP2基因mRNA和蛋白的抑制效果；噻唑蓝(MTT)比色法观察抑制MRP2基因表达后,HepG2/ADM细胞对化疗药物的敏感性变化,并计算各药物的半数抑制浓度(IC50).结果 HepG2/ADM对ADM、5-氟尿嘧啶(5-Fu)、长春新碱和奥沙利铂的IC50值分别为0.3204、3.8002、0.2014和0.1221；siRNA明显抑制了HepG2/ADM细胞MRP2基因mRNA和蛋白的表达(P＜0.05)；转染MRP2-siRNA后,HepG2/ADM细胞对ADM、5-Fu、长春新碱和奥沙利铂的IC50值明显减小,分别为0.1023、1.4417、0.0452和0.0268.结论用siRNA沉默MRP2基因能够逆转HepG2/ADM细胞对化疗药物的耐药性,MRP2基因与HepG2/ADM肝癌细胞的多药耐药性相关,可通过沉默MRP2基因提高耐药细胞对化疗药物的敏感性.
목적 검측소간우RNA(siRNA)대간암내약세포주HepG2/아매소(ADM)다약내약상관단백기인2(MRP2)급기단백산물적억제작용,관찰대다약내약성적영향.방법 사용농도제도법제작HepG2내ADM세포주(HepG2/ADM),ADM농도종0.1～2.0 m/L:설계합성침대MRP2적siRNA소편단,전염HepG2/ADM내약세포,24 h후통과실시형광정량역전록-취합매련반응(RT-qPCR)화Western blot검측siRNA대MRP2기인mRNA화단백적억제효과；새서람(MTT)비색법관찰억제MRP2기인표체후,HepG2/ADM세포대화료약물적민감성변화,병계산각약물적반수억제농도(IC50).결과 HepG2/ADM대ADM、5-불뇨밀정(5-Fu)、장춘신감화오사리박적IC50치분별위0.3204、3.8002、0.2014화0.1221；siRNA명현억제료HepG2/ADM세포MRP2기인mRNA화단백적표체(P＜0.05)；전염MRP2-siRNA후,HepG2/ADM세포대ADM、5-Fu、장춘신감화오사리박적IC50치명현감소,분별위0.1023、1.4417、0.0452화0.0268.결론 용siRNA침묵MRP2기인능구역전HepG2/ADM세포대화료약물적내약성,MRP2기인여HepG2/ADM간암세포적다약내약성상관,가통과침묵MRP2기인제고내약세포대화료약물적민감성.
Objective To investigate the suppression of multidrug resistance-associated proteins 2 (MRP2) and the protein product induced by small interfering RNA (siRNA) in the multidrug-resistant (MDR) hepatocellular carcinoma (HCC) cell line HepG2/adriamycin (ADM) and the effect on MDR.Methods MDR HepG2/ADM cells were developed by exposing parental cells to stepwise increasing concentrations of ADM from 0.1 to 2.0 mg/L.MRP2 targeted small interfering RNA fragments were designed and synthesized,and transfected into MDR HepG2/ADM cells.The suppression of MRP2 and the protein product were detected by using real-time reverse transcription quantitative poiymerase chain reaction (RT-qPCR) and Western blotting at 24 h after transfection.Methyl thiazol tetrazolium (MTT) assay was used to determine drug sensitivity of HepG2/ADM cells before and after transfection based on the results of IC50.Results MTT assay showed that the IC50 values of HepG2/ADM against ADM,5-fluorouracil,vincristine,and oxaliplatin were 0.3204,3.8002,0.2014 and 0.1221 respectively.siRNA transfection significantly inhibited the expressiou of MRP2 mRNA and protein in HepG2/ADM cells ( P ＜ 0.05).After MRP2-siRNA transfection,the IC50 values of HepG2/ADM against ADM,5-fluorouracil,vincristine,and oxaliplatin were decreased significantly (0.1023,1.4417,0.0452 and 0.0268,respectively).Conclusion Silencing MRP2 genes by siRNA can reverse MDR of HepG2/ADM cells.MRP2 is closely related to MDR of HepG2/ADM HCC cells and silencing MRP2 might improve the sensitivity of resistant cells to chemotherapeutic drugs.