摘要
目的:探讨程序性细胞死亡蛋白4(programmed cell death protein 4,PDCD4)对人类慢性粒细胞白血病细胞系K562细胞活力、凋亡以及基因表达变化的影响。方法:通过慢病毒感染的方法在K562细胞系中过表达PDCD4基因并筛选获得稳定表达的细胞系;采用RT-qPCR和Western blot分别检测PDCD4 mRNA和蛋白的表达水平;CCK-8法检测细胞活力;流式细胞术检测细胞周期和细胞凋亡情况;应用转录组测序(RNA sequencing,RNA-seq)分析PDCD4过表达组和对照组的基因表达差异及其相关的功能与信号通路,RT-qPCR验证测序结果的准确性。结果:成功建立了稳定过表达PDCD4的K562细胞系;PDCD4过表达可显著抑制K562细胞的活力,引起细胞周期阻滞并促进阿糖胞苷诱导的细胞凋亡(P<0.05);RNA-seq结果显示,在K562细胞中过表达PDCD4可引起大量的基因表达发生变化,表达明显差异的基因共有394个,其中上调16个,下调378个,主要涉及细胞周期、凋亡、细胞自噬和代谢等方面,选取的6个基因的RT-qPCR检测结果和RNA-seq的结果一致。结论:PDCD4可抑制K562细胞活力、阻滞细胞周期进展、促进细胞凋亡;RNA-seq确定了PDCD4对K562细胞基因表达谱的变化。
AIM:To investigate the effect of programmed cell death protein 4(PDCD4)on viability,apopto-sis and the gene expression of human chronic myeloid leukemia cell line K562.METHODS:PDCD4 was over-expressed by lentivirus infection in K562 cells,and the cells stably expressing PDCD4 were selected by puromycin.The expression of PDCD4 at mRNA and protein levels were determined by RT-qPCR and Western blot.The viability of K562 cells was measured by CCK-8 assay,cell cycle and apoptosis were analyzed by flow cytometry.In addition,RNA sequencing(RNA-seq)was used to screen the alteration of gene expression profile after PDCD4 over-expression to identify the down-stream genes regulated by PDCD4 in K562 cells,and in which function and pathway the differential genes were involved.The expression of differential genes was detected by RT-qPCR to verify the accuracy of the RNA-seq results.RESULTS:We established K562 cells with stable over-expression of PDCD4,and found that over-expression of PDCD4 significantly inhibited the cell viability,induced cell cycle arrest,and promoted cytosine arabinoside induced apoptosis.The results from RNA-seq found that there were 394 differentially expressed genes between PDCD4 over-expressed K562 cells and control K562 cells.Among them,16 genes expression were up-regulated and 378 genes expression were down-regulated,which mainly involved in cell cycle,apoptosis,autophagy and metabolism,etc.6 differential genes expression were de-tected by RT-qPCR,which were consistent with the RNA-seq results.CONCLUSION:PDCD4 inhibits viability,in-duces cell cycle arrest and promotes cell apoptosis in K562 cells.Moreover,we confirmed the differentially expressed genes after PDCD4 over-expression in K562 cells.
作者
龙馨妍
何泽诏
彭鹏
李涛
张慧慧
袁仕善
张霞
LONG Xin-yan;HE Ze-zhao;PENG Peng;LI Tao;ZHANG Hui-hui;YUAN Shi-shan;ZHANG Xia(Department of Immunology,School of Medicine,Hunan Normal University,Changsha 410013,China;Department of Biochemistry,School of Medicine,Hunan Normal University,Changsha 410013,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第7期1210-1218,共9页
Chinese Journal of Pathophysiology
基金
湖南省自然科学基金资助项目(No.2018JJ3365)
湖南省教育厅重点项目(No.20A308)
湖南省卫生健康委一般资助课题(No.202102071747)。
