构建COX2基因慢病毒载体对肺腺癌A549细胞增殖与凋亡的影响#br#

目的 构建环氧化酶2（COX2）基因慢病毒表达载体并观察其对非小细胞肺癌A549细胞增殖与凋亡的影响。方法 针对COX2基因的不同片段设计3对shRNA的寡核苷酸片段并克隆至慢病毒载体GV248中，构建并筛选最佳抑制效率的靶向COX2基因的慢病毒载体COX2 shRNA。选取对数生长期A549细胞进行慢病毒转染，其中靶向COX2基因的shRNA载体转染A549细胞为shRNA组，以空载体质粒转染作为阴性对照组（NC）及不转染质粒的A549细胞为空白对照组（CON），采用实时荧光定量PCR和Western blotting测定COX2基因和蛋白的表达。分别采用MTT法和流式细胞仪检测干扰COX2表达对A549细胞增殖、凋亡能力的影响。结果 成功构建靶向COX2基因的慢病毒载体；稳定转染A549细胞后，COX2基因表达下降，其中以COX2 shRNA1干扰效率最为显著；shRNA组中shRNA1转染的A549细胞COX2 mRNA水平为0.17±0.06，低于NC组的081±011和CON组的109±0.07，差异有统计学意义（P＜0.05）。shRNA组细胞增殖活性低于NC组和CON组，shRNA组转染5 d的吸光值为0.976±0.016，低于NC组的1.557±0.015和CON组的1880±0034，差异有统计学意义（P＜0.05）。shRNA组细胞凋亡率为（6.28±0.31）％，高于NC组的（3.08±0.05）％和CON组的（3.01±0.31）％，差异有统计学意义（P＜0.05）。结论 成功构建靶向COX2基因的慢病毒表达载体，干扰COX2表达可抑制人肺腺癌A549细胞的增殖并促进细胞凋亡。

Construction of lentivirus vector carrying COX2 gene and its effect on proliferation and apoptosis of non small cell lung cancer A549 cells

ObjectiveTo construct a recombinant short hairpin RNA （shRNA） lentiviral vector of cyclooxygenase 2 （COX2） gene， and investigate its effects on proliferation and apoptosis of non small cell lung cancer （NSCLC）. MethodsThree oligonucleotides targeting COX2 gene were synthesized and cloned into lentivirus vector GV248. The lentivirus vector COX2 shRNA targeted to COX2 gene was constructed and screened for the best inhibition efficiency. The human NSCLC A549 cells were selected for lentivirus transfection during the logarithmic growth period. Specially， shRNA vector targeting COX2 gene was transfected into A549 cells as shRNA group， and empty vector plasmid was transfected as negative control （NC） group. A549 cell without transfection plasmid were selected as blank control group （CON）. COX2 expressions were assessed by real time PCR and Western blotting. The effects of interfering COX2 expression on the proliferation and apoptosis of A549 cells were detected by MTT and flow cytometry. ResultsThe recombinant lentivirus vector targeting COX2 was constructed successfully. Especially COX2 shRNA1 showed the best transfection efficiency. In shRNA group， the level of COX2 mRNA in shRNA1 transfected A549 cells was 017±006， lower than 081±011 of NC group and 109±007 of CON group （P＜0.05）. The proliferative activity of shRNA group was lower than those of NC group and CON group. For example， the absorbance value at 5 d in shRNA group was 0976±0016， which was lower than 1.557±0.015 of NC group and 1.880±0.034 of CON group （P＜0.05）. The apoptotic rate of shRNA group was （6.28±0.31）％， which was higher than （3.08±0.05）％ of NC group and （3.01±0.31）％ of CON group， and the difference was statistically significant （P＜0.05）. ConclusionThe recombinant lentivirus shRNA vector target COX2， with best transfection efficiency， is constructed successfully. RNAi mediated silencing of the COX2 gene can inhibit the proliferation of A549 cells and promote its cell apoptosis.