Akt2特异性siRNA对人肺癌细胞化疗敏感性及耐药性的影响

Effects of Akt2-siRNA on chemotherapeutic sensitivity and drug resistance in human lung cancer cells

目的探讨癌基因蛋白质v-akt（Akt2）特异性siRNA对人肺癌细胞株NCI—H446细胞顺铂化疗敏感性的影响及对耐药蛋白的调控作用。方法构建针对Akt2基因mRNA的siRNA，利用脂质体转染肺癌细胞NCI-H446，反转录-PCR检测Akt2-mRNA的表达，免疫细胞化学法检测肺耐药相关蛋白（LRP）及P糖蛋白（P-gP）的表达。以顺铂分别作用于对照组、未转染组、阴性质粒组及转染组细胞24h后，噻唑蓝（MTr）比色法检测细胞的增殖，流式细胞仪检测细胞的凋亡。结果转染Akt2-siRNA后，NCI-H446细胞Akt2-mRNA表达明显降低，转染组肺癌细胞LRP及P-gP表达水平显著低于未转染组（P〈0．01）。细胞的增殖率从（60．2±2．8）％下降到（34．7±2．6）％（P〈0．01），凋亡率从（19．3±1．6）％上升到（38．8±1．2）％（P〈0．01）。结论Akt2特异性siRNA可明显下调Akt2的表达，提高NCI—H446细胞对化疗的敏感性；它可能是通过下调LRP和P—gp蛋白的表达，部分逆转NCI-H446细胞对顺铂的耐药性。

Objective To explore the effects of oncogene protein v-akt-siRNA on the sensitivity of human lung cancer cell line NCI-H446 to cisplatin and drug resistance proteins in human lung cancer ceils. Methods The small interfering siRNA expression vector targeting Akt2 gene （siAkt2） was constructed. And the NCI-H446 cells were transfected with negative control vector or siRNA vector. The expressions of Akt2-mRNA and lung resistance-related protein （LRP） and P-glycoprotein （P-gp） were detected by reverse transcription-polymerase chain reaction and immunocytochemistry respectively. NCI- H446 and transfected cells were treated by cisplatin for 24 h. The cell proliferation was measured by 3- （4, 5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium （MTT） assay and cell apoptotic rate detected by flow cytometry. Results Akt2-mRNA decreased significantly in the transfected NCI-H446 cells versus the non- transfection group. And the expressions of LRP and P-gp proteins decreased significantly in the transfection group versus the control group （P 〈 0. 01 ）. The cell proliferation rate decreased from （60. 2 ± 2. 8） % to （34.7 ±2.6）% （P〈0.01）. The cell apoptotic rate increased from （19.3 ±1.6）% to （38.8 ±1.2）% after a therapy of cisplatin （ P 〈 0. 01 ）. Conclusion The siRNA targeting Akt2 can decrease the Akt2 expression, increase the chemotherapeutic sensitivity to cisplatin and partially reverse the cisplatin resistance of NCI-H446. The mechanism may be through the lowered expressions of LRP and P-gp.