NANOG基因对急性淋巴细胞白血病细胞增殖及细胞周期的影响

Effects of NANOG Expression on Proliferation and Cell Cycle of Acute Lymphoblastic Leukemia Cells

目的 探讨NANOG基因在人类急性淋巴细胞白血病（ALL）细胞株中的表达,并研究下调该基因表达对ALL细胞增殖和细胞周期的影响以及可能机制.方法 选取ALL的MOLT-4、CCRF-HSB2、Jurkat 3种细胞系为研究对象.利用逆转录-聚合酶链反应（RT-PCR）及Western blot技术检测MOLT-4、CCRF-HSB2、J urkat细胞系中NANOG的表达情况.通过构建携带NANOG基因的特异性shRNA的慢病毒载体包装病毒颗粒（pLB-shNANOG-1和pLB-shNANOG-2）,该病毒感染MOLT-4细胞后,经分选获得稳定表达株作为实验组：shNANOG-1组和shNANOG-2组;以空质粒（pLB-sh control）感染的细胞和正常MOLT-4细胞分别作为阴性对照组和空白对照组.在基因及蛋白水平下,检测各组NANOG的干扰效率.采用CCK-8法及流式细胞仪检测实验组和对照组细胞增殖能力及细胞周期的变化,并利用实时定量PCR技术检测p53通路相关基因的表达变化.结果 包装病毒颗粒超速离心后病毒滴度达（1.83～3.12）×108 IU/mL.2种shRNA可有效下调病毒感染MOLT-4细胞的NANOG基因及蛋白的表达.CCK-8法检测显示shNANOG-1及shNANOG-2组细胞在不同时间点其光密度值（OD）较空白对照组及阴性对照组下降,在培养72 h后最为明显,实验组与对照组比较,差异均有统计学意义（P＜0.001）.细胞周期检测显示,shNANOG-1及shNANOG-2组细胞在G0/G1期细胞比例较空白以对照组及阴性对照组高（P＜0.001）,但两组细胞在S期细胞比例较空白对照组及阴性对照组低（P＜0.01）,而在G2/M期细胞比例在各组间差异无统计学意义（P＞0.05）.实时定量PCR法证实shNANOG-1及shNANOG-2组细胞中TP53及CDKN1A基因表达上调,而MDM2及CCND1基因表达下调,实验组与对照组相比差异均有统计学意义（P＜0.05）.结论 NANOG在部分人类ALL细胞系中有表达,下调NANOG后可通过MDM2-TP53-CDKN1A通路抑制白血病细胞的增殖,并使细胞阻滞于G0/G1期.

Objective To explore the expression of NANOG gene in acute lymphoblastic leukemia （ALL） cell lines and the effect of down-regulation of NANOG on the proliferation and cell cycle of ALL cells.Methods Three kinds of ALL cell line MOLT-4,CCRF-HSB2 and Jurkat were chosen as study object.The expression of NANOG was detected by reverse transcription-polymerase chain reaction （RTPCR） and Western blot in MOLT-4,CCRF-HSB2 and Jurkat cells.The lentiviral vector （pLB-shNANOG-1 and pLB-shNANOG-2） carrying NANOG specific shRNA were constructed.After infection of MOLT-4 cells with the lentivirus constructs,green fluorescent protein positive cells were harvested by flow cytometry as interference group.With empty plasmid （pLB-sh control） infected MOLT-4 cells and normal cells were used as wild-type group and control group.The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot.Cell proliferation was evaluated using CCK-8 cell counting kit.Cell cycle was analyzed by flow cytometry.The expression of p53-related gene was detected by real-time quantitative PCR.Results The virus titers were （1.83-3.12）×108 IU/mL.Each interfering sequence cloud stably down-regulate the expression of NANOG.Analysis of cell proliferation indicated that the MOLT-4 cells expressing NANOG shRNA grew significantly slower than wild-type cells and control cells,and this difference became more obvious after 72 hours （P＜0.001）.Cell cycle analysis indicated that the percentage of S-phase cells in wild-type or control group cells was obviously increased when compared to MOLT-4 cells expressing NANOG shRNAs （P＜0.01）.The percentage of MOLT-4 cells expressing NANOG shRNAs in the G0/G1-phase was higher than in wild-type cells and control cells （P＜0.001）,while the percentage of cells in the G2/M-phase was not statistical different among these cells （P＞0.05）.The expression of gene TP53 and CDKN1A were increased in groups of shNANOG-1 and shNANOG-2 than that of groups of control and wild-type （P＜0.05）.But the expression of gene MDM2 and CCND1 was decreased （P＜0.05）.Conclusions NANOG is expressed in some human ALL cell lines.Down-regulation of NANOG could inhibit cell proliferation and arrest in the cell cycle of G0/G1 phase through a MDM2-TP53 CDKN1A pathway in leukemic cells.