利用新型载体运转MDR1 siRNA以逆转胰腺癌化疗耐药性的研究

Reversal of chemoresistance in pancreatic cancer line by delivering an MDR1 small interfering RNA (siRNA) by a adenoassociated Vector

目的:以新型载体运转MDR1 siRNA导入胰腺癌细胞株后,观察其多药物耐药基因(MDR1)的表达及其对胰腺癌化疗耐药性的影响。方法:采用RNA干扰技术,构建可用于包装成自我复制rAAV病毒载体的质粒,以运转不同长度的MDR1 siRNA。采用实时PCR检测MDR1 mRNA的表达,Western印迹法检测总P-糖基化蛋白(P-gp)的表达水平,用流式细胞仪检测细胞表面的P-gp表达,并从不同层面检测胰腺癌细胞株P-gp表达的抑制率,从而筛选出最佳的质粒载体。采用MTT检测IC50值,进行各载体逆转胰腺癌细胞株化疗耐药性的研究。结果:拟用于构建自我复制rAAV病毒载体的25-mer MDR1 siRNA质粒能有效地将目的基因MDR1 siRNA导入胰腺癌细胞株,并予稳定的表达,发挥作用。导入胰腺癌细胞株的MDR1 siRNA能有效地在mRNA水平上沉默MDR1基因,显著抑制其表达,使其蛋白产物P-gp的表达明显减少,及其在细胞膜上的数量显著降低。结果能有效、显著地逆转胰腺癌细胞株的化疗耐药性,转染前SW1990/ADM细胞对ADM的IC50值约是转染后的50倍。结论:采用可用于构建新型sc-rAAV载体的质粒作为投递siRNA的工具,并选择MDR1为研究靶点,通过实验证实该策略在逆转胰腺癌化疗耐药性中的有效性。

Objective The inhibitory effect of siRNA has been shown to counteract the resistance to chemotherapy by suppressing the expression of MDR-1 gene-encoded P-gp in pancreatic cancer.Our research involues an investigation of the reversal of chemoresistance a pancreatic cancer cell line by delivering an MDR1 siRNA by self-complementary AAV（scAAV）,a new style vector.Methods The sequences of the hairpin siRNAs for anti-MDR1 25-mer and 28-mer,mismatch MDR1,targeting MDR1 mRNA at nt 1545-1565 or their extensions were synthesized and inserted into BamHI-HindIII linearized pSilencer 2.1-U6 hygro vector.The plasmid and the control plasmid were stably transfected into human pancreatic cancer cell lines SW1990/ADM and SW1990.RNA extraction and real-time RT-PCR were performed to investigate the effect of vectors in the mRNA level.Total cellular P-glycoprotein by was detected Western blot.P-glycoprotein expression on cell surface was measured by flow cytometry.MTT was applied to the chemo-sensitivity test of chemotherapeutic agents on tumor cell lines.Results The results showed correlated degrees of reduction in the MDR1 mRNA level and total P-glycoprotein level in groups of 25-mer and 28-mer.The 25-mer group was found to be more effective than the latter.In the control groups,the expressed levels of MDR1 mRNA and P-glycoprotein were similar to those of untreated controls.And the result of MTT showed ADM-response profiles of the 25-mer group to be significantly left-shifted,with IC50 values of 1.5.Conclusions Based on this experience,we extrapolate that the technique of siRNA and this type of vector may be a valuable tool in therapy of pancreatic cancer.