Pirh2 shRNA增强肺腺癌细胞A549对顺铂敏感性的研究

Effect of Pirh2 shRNA on Chemosensitivity of Lung Adencarcinoma Cell Line A549 to Cisplatin

目的观察短发夹状RNA沉默泛素连接酶pirh2(P53 induced RING-H2 protein)基因表达后,肺腺癌细胞A549对顺铂敏感性及肺耐药蛋白(lung resistance-related protein,LRP)表达的变化。方法构建靶向pirh2基因的短发夹状RNA(psiRNA-Pirh2)及阴性对照RNA(psiRNA-Con),并转染A549细胞,实时定量PCR和免疫印迹法检测A549细胞中pirh2基因和蛋白的表达变化。设立顺铂组(5μg/ml)、psiRNA-Pirh2转染组和顺铂(5μg/ml)+psiRNA-Pirh2组,CCK-8法检测A549细胞增殖能力,流式细胞术检测细胞周期,免疫印迹法检测肺耐药蛋白的表达,以正常A549细胞为对照。结果psiRNA-Pirh2特异性抑制了A549细胞pirh2 mRNA和蛋白质的表达(均P<0.05)。顺铂及psiRNA-Pirh2组A549细胞增殖能力分别为正常对照组的(46.82±6.14)%和(37.45±6.83)%,均P<0.05,但均高于二者联合组[(28.24±6.49)%],且联合组细胞凋亡率最高,达(19.8±5.1)%,S期细胞最少,为(15.5±1.6)%(P<0.05)。顺铂处理组A549细胞LRP表达较正常对照组增强(P<0.05),但联用psiRNA-Pirh2后,LRP蛋白水平有所下降(P<0.05)。结论靶向pirh2的shRNA可特异性降低A549细胞中pirh2的表达,抑制A549细胞增殖,使其阻滞在G2/M期,诱导凋亡,下调肺耐药蛋白的表达并增强其对顺铂的化疗敏感性。

Objective To investigate the effects of pirh2 short hairpin RNA （shRNA） on the pirh2 expression, cell proliferation, apoptosis and chemosensitivity of lung adencarcinoma cell line A549 to cisplatin. Methods Two shRNA plasmids were constructed, one of which was designed targeting pirh2 （named psiRNA-Pirh2）, and the other targeting nothing as negativecontrol （named psiRNA-Con）. Both of them were transfected into A549 cells by Lipofectamine^TM 2000 respectively. Real-time PCR and Western-blotting were applied to detect the expression level of pirh2. Cisplatin （5 μg/ml） was added into the normal A549 cells as well as that transfected psiRNA-Pirh2. The ability of cell proliferation was analyzed by cell counting kit-8 （CCK- 8） assay. Flow cytometry was used to examine the cell cycles distribution. The expression level of lung resistance-related protein （LRP） was detected by Western-blotting. Results The expression levels of pirh2 mRNA and protein were obviously lower in psiRNA-Pirh2-transfected A549 cells than that of the psiRNA-Con-transfected or untransfected （P〈0.05）. A549 cells treated with both cisplatin and psiRNA-Pirh2 grew slowest and the percentage of cells in S phase was lowest, on the contrary, the percentage of cells in G2/M phase and apoptotic rate were highest. The LRP protein expression in A549 cells only treated with cispaltin was higher than that of normal A549 cells, but when combined with psiRNA-Pirh2, LRP protein expression was down-regulated （P〈0.05）. Conclusion Sequence-specific shRNA targeting pirh2 could down-regulate the expression of pirh2 gene and suppress effectively A549 cells growth. In addition, it attenuates the level of LRP protein, therefore sensitizes A549 cells to cisplatin.