小干扰RNA沉默干扰素调节因子-8对缺氧复氧后肺泡上皮细胞凋亡的影响

Effect of small interfering RNA silencing interferon regulatory factor-8 on apoptosis of alveolar epithelial cells after hypoxia-reoxygenation

目的观察小干扰RNA(siRNA)靶向沉默干扰素调节因子-8(IRF-8)对缺氧复氧后肺泡上皮细胞凋亡的影响。方法将IRF-8 siRNA、Negative control(NC)siRNA通过脂质转染试剂转染至肺泡上皮细胞株[L2(购自中国上海拜利生物科技有限公司)],将细胞分为4组,si-IRF-8组,转染含有IRF-8 si-RNA的细胞组;质粒对照组,转染Negative control(NC)siRNA的细胞组;正常对照组,无转染质粒,完全正常培养;阴性对照组,无质粒转染,给与缺氧复氧干预。通过蛋白质印迹法(Western blot)检测肺泡上皮细胞L2细胞株转染后凋亡相关分子标志物:活化的半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3);聚合酶链反应(PCR)检测Caspase-3、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、B细胞淋巴瘤/白血病-2(bcl-2)的转录水平变化。组间比较采用独立样本t检验。结果转染IRF-8 siRNA后,L2细胞内IRF-8相对表达水平明显下调,差异有统计学意义[si-RNA组(0.146±0.009)对比si-NC组(1.001±0.055),t=15.566,P<0.01]。经过缺氧复氧干预后L2细胞株si-IRF-8组Caspase-3、bax水平(1.877±0.250、1.989±0.145)较si-NC组(3.681±0.292、4.636±1.261)明显降低,差异有统计学意义(t=-4.686、-5.110,P<0.01),bcl-2水平在L2细胞的si-IRF-8组(0.837±0.858)较si-NC(0.179±0.533)组显著升高,差异有统计学意义(t=15.955,P<0.01);Western blot检测cleaved Caspase-3蛋白的表达水平(0.257±0.051)较质粒对照组(0.485±0.751)明显下调,差异有统计学意义(t=6.205,P<0.01)。结论IRF-8 si-RNA能特异性下调肺泡上皮细胞L2细胞株中IRF-8的表达,并显著降低缺氧复氧干预后细胞的凋亡水平,其机制可能与下调促凋亡相关蛋白bax、Caspase-3及上调抗凋亡相关蛋白bcl-2的转录过程相关。

Objective To observe the effect of small interfering RNA(siRNA)targeting silencing interferon regulatory factor-8(IRF-8)on apoptosis of alveolar epithelial cells after hypoxia-reoxygenation.Methods The L2 cells were divided into two groups:IRF-8 siRNA transfection group(si-IRF-8-RNA group)and negative control siRNA transfection group(si-NC group).After 72 h of transfection,the cells were cultured under hypoxia for 2 h and then cultured normally for 2 h.The expression of cleaved Caspase-3,an apoptosis-related molecular marker,was detected by Western blotting.The transcriptional levels of Caspase-3,B cell lymphoma/leukemia-2 associated X protein(bax),and B-lymphoma-2(bcl-2)genes were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results The expression of IRF-8 obviously decreased in si-IRF-8-RNA group(0.146±0.009)compared to si-NC group(1.001±0.055),t=15.566,P<0.01.After hypoxia-reoxygenation intervention,the levels of Caspase-3 and bax in si-IRF-8 group(1.877±0.250,1.989±0.145)were significantly lower than those in si-NC group(3.681±0.292,4.636±1.261)and the difference was statistically significant(t=-4.686,-5.110,P<0.01).On the contrary,the level of bcl-2 in the si-IRF-8 group of L2 cells(0.837±0.858)was significantly higher than that in the si-NC group(0.179±0.533)(t=15.955,P<0.01).Western blotting showed the expression level of cleaved Caspase-3 protein in si-IRF-8 group(0.257±0.051)was significantly lower than that in si-NC group(0.485±0.751)and the difference was statistically significant(t=6.205,P<0.01).Conclusion RNAi vectors of IRF-8 can specifically down-regulate the expression of IRF-8 in alveolar epithelial cells L2 and significantly reduce the apoptotic level of cells after hypoxia-reoxygenation intervention.The mechanism may be related to the down-regulation of apoptotic-related proteins bax,Caspase-3 and up-regulation of the transcription of anti-apoptotic-related proteins bcl-2.