靶向MRP1基因的shRNA稳定逆转肺癌的多药耐药性  

Stable reversal of multidrug resistance in A549/DDP cells by shRNA targeting MRP1gene

作　　者：邵淑丽[1] 崔婷婷[1] 贾红双[1] 谢振丽[1] 张伟伟[1] 刘迁[1] 陈薇薇[1] 李爽[1] 陈丽[1] 

机构地区：[1]齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔161006

出　　处：《中国病理生理杂志》2011年第12期2265-2269,共5页Chinese Journal of Pathophysiology

基　　金：黑龙江省自然科学基金资助项目(No.C200624);黑龙江省教育厅科学技术项目(No.11511447No.12511611)

摘　　要：目的:利用短发夹RNA(shRNA)表达载体逆转肺癌细胞株(A549/DDP)的多药耐药性。方法:构建2个多药耐药相关蛋白1(MRP1)基因特异的shRNA表达载体pSilencer 2.1-U6 neo-MRP1,稳定电转染A549/DDP细胞,实时荧光定量PCR分析MRP1 mRNA的表达,免疫荧光检测细胞MRP1蛋白的表达,流式细胞术检测细胞内罗丹明123(Rho123)的潴留情况。MTT法检测细胞活力。结果:成功构建了shRNA表达载体pSilencer 2.1-U6 neo-MRP1,稳定转染sh-MRP1-2.1-1和sh-MRP1-2.1-2后,A549/DDP细胞MRP1 mRNA和蛋白表达均显著降低,细胞内Rho123相对荧光强度由16.93%±0.58%分别升高至89.02%±0.59%和82.56%±1.37%;A549/DDP亲本细胞顺铂的IC50分别由(101.45±0.64)μmol/L降至(38.06±0.05)μmol/L和(53.72±0.36)μmol/L,5-氟尿嘧啶的IC50分别由(263.20±2.00)μmol/L降至(98.82±1.16)μmol/L和(141.81±0.49)μmol/L。结论:shRNA干扰表达载体pSilencer 2.1-U6 neo-RMP1能够稳定、持久地抑制MRP1基因,有效地逆转了A549/DDP细胞的多药耐药性。AIM： To reverse multidrug resistance（MDR） of A549/DDP cells with short hairpin RNA（shRNA） expression vectors.METHODS： Two multidrug resistance-associated protein 1（MRP1） gene-specific shRNA expression plasmids pSilencer 2.1-U6 neo-MRP1 were constructed and introduced into A549/DDP cells.MRP1 mRNA was assayed by real-time fluorescent quantitative PCR.The MRP1 function was determined by rhodamine 123（Rho123） retention and the protein expression of MRP1 was detected by immunofluorescent staining.The viability of A549/DDP cells was evaluated by MTT method.RESULTS： MRP1 shRNA expression plasmids were successfully constructed.The expression of MRP1 at mRNA and protein levels was significantly decreased after sh-MRP1-2.1-1 and sh-MRP1-2.1-2 were transfected into A549/DDP cells.The intracellular accumulation of Rho123 significantly increased from（16.93±0.58）% to（89.02±0.59）% and（82.56±1.37）%.IC50 of cisplatin were decreased from（101.45±0.64） μmol/L to（38.06±0.05） μmol/L and（53.72±0.36） μmol/L.IC50 of 5-fluorouracil were decreased from（263.20±2.00） μmol/L to（98.82±1.16） μmol/L and（141.81±0.49） μmol/L.CONCLUSION： The shRNA expression plasmid pSilencer 2.1-U6 neo-MRP1 can stably and permanently inhibit MRP1 gene.The sensitivity of A549/DDP cells to drug is reversed.

关 键 词：短发夹RNA 多药耐药 多药耐药相关蛋白1 RNA干扰 A549/DDP细胞 

分 类 号：R734.2[医药卫生—肿瘤]