下调生长阻滞和DNA损伤诱导蛋白45β表达对PC9肺腺癌细胞的影响  

Effect of down-regulation of growth arrest and DNA damage inducible protein 45β on PC9 lung adenocarcinoma cells

作　　者：胡皓[1] 阙凯琳 彭昊[1] 刘佳[1] 韩宸 张娜[1] 侯涛[1] 胡春宏[1] 马进安[1] HU Hao;QUE Kailin;PENG Hao;LIUJia;HAN Cheng;ZHANG Na;HOU Tao;HU Chunhong;MA Jin'an(Department of Oncology,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院肿瘤科,长沙410011

出　　处：《中南大学学报（医学版）》2018年第11期1209-1215,共7页Journal of Central South University ：Medical Science

摘　　要：目的:探讨下调生长阻滞和DNA损伤诱导蛋白45β(growth arrest and DNA damage inducible protein 45β,GADD45β)表达对PC9肺腺癌细胞及吉非替尼敏感性的影响。方法:设计并合成GADD45β基因小干扰RNA(GADD45β-small interfering RNA,GADD45β-siRNA)序列,通过慢病毒介导将GADD45β-siRNA转入PC9肺腺癌细胞中,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western印迹检测转染前后PC9肺腺癌细胞GADD45β的mRNA及蛋白水平,采用膜联蛋白V(annexin V)-别藻蓝蛋白(allophycocyanin,APC)双染流式细胞法检测转染后细胞凋亡水平;通过流式细胞术检测转染后细胞内DNA含量,计算转染后细胞各周期时相百分率,分析转染对细胞生长周期的影响;通过计数克隆形成数检测RNA干扰对细胞成瘤能力的影响;采用MTT法检测PC9肺腺癌细胞的吉非替尼半数抑制浓度IC50。结果:筛选出5'-AAATCCACTTCACGCTCAT-3'为GADD45β基因RNA干扰的有效序列。转染GADD45β-siRNA 48 h后,qRT-PCR和Western印迹结果显示PC9肺腺癌细胞GADD45β的mRNA和蛋白表达水平明显下调(均P<0.05),细胞凋亡率明显增加(P<0.05),且成瘤克隆数明显减少(P<0.05);PC9肺腺癌细胞位于S期及G2/M期细胞增多(P<0.05),吉非替尼的IC50明显下降(P<0.05)。结论:PC9肺腺癌细胞转染GADD45β-siRNA后,能成功下调GADD45β基因的mRNA和蛋白表达;下调GADD45β表达可降低PC9肺腺癌细胞的克隆形成能力,促进细胞凋亡;下调GADD45β表达可明显提高PC9肺腺癌细胞对吉非替尼的敏感性。Objective:To explore the effect of down-regulation of growth arrest and DNA damage inducible protein 45β(GADD45β) on the PC9lung adenocarcinoma cells.Methods:GADD45β gene siRNA sequence was designed and synthesized,which was transfected into PC9 lung adenocarcinoma cells through lentivirus transfection.Quantitative real-time PCR (qRT-PCR)and Western blot are used to examine the mRNA and protein levels of GADD45β in PC9 cells before and after the transfection.Annexin V-allophycocyanin (APC)double-staining flow cytometry was used to detect the apoptosis level after the transfection.The intracellular DNA content after transfection was detected by flow cytometry.The percentage of the cells at each period of cell cycle was calculated,and the effect of RNA interference on the cell growth were analyzed. The effects of RNA interference on the tumor-formation ability of ceils were tested by counting the number of clones.MTT assay was used to test the half maximal inhibitory concentration (IC50)of PC9 cells for gefitinib. Results:The 5'-AAATCCACTTCACGCTCAT-3' sequence was identified as the effective sequence for GADD45β gene RNA interference.The mRNA and protein expression levels of GADD45β were markedly decreased (both P<0.05) at 48h after transfection of GADD45β-siRNA,which resulted in the increased apoptosis rate (P<0.05),decreased tumor clone number (P<0.05)and increased percentage of PC9 cell at the S stage and G2/M stage (P<0.05).The IC50 for gefitinib was decreased obviously (P<0.05). Conclusion:Down-regulation of GADD45β can reduce the colony-forming ability of PC9 cells, promote the cell apoptosis,and enhance the sensitivity of PC9 cells to gefitinib.

关 键 词：肺腺癌 生长阻滞和DNA损伤诱导蛋白45β RNA干扰 吉非替尼 半抑制浓度 

分 类 号：R734.2[医药卫生—肿瘤]