NANOG基因表达下调对急性T淋巴细胞白血病细胞凋亡的影响

Effects of NANOG gene down-regulation on the apoptosis of T-cell acute lymphoblastic leukemia cells

目的探讨NANOG基因在人急性T淋巴细胞白血病（WALL）细胞株中的表达及下调该基因表达对细胞凋亡的影响及可能机制。方法利用RT-PCR及Westernblot方法检测T-ALL细胞系MOLT-4、CCRF—HSB2、Jurkat细胞NANOG基因表达情况。通过构建携带NANOG特异性shRNA的慢病毒载体包装病毒颗粒，感染MOLT-4细胞后经分选获得稳定表达株，并在基因及蛋白水平检测NANOG干扰效率。用流式细胞术检测各组细胞早期凋亡（Annexin VV7-AAD-）及晚期凋亡（Annexin V＋/7-AAD＋）率，并利用实时定量PCR技术检测凋亡相关基因的表达情况。结果MOLT-4和CCRF—HSB2细胞NANOGmRNA及蛋白表达阳性。构建慢病毒干扰质粒pLB．shNANOG-1、pLB—shNANOG-2及pLB．shRNA对照，包装病毒颗粒超速离心后病毒滴度达（1．83～3．12）×10^8IU／ml。病毒感染MOLT-4细胞后经实时定量PCR及Wesmmblot方法证实两种shRNA可有效下调NANOGmRNA及蛋白的表达。流式细胞术检测显示shNANOG．1及shNANOG。2组细胞早期凋亡率分别为（8．06±1．61）％及（5．67±1．59）％，较MOLT-4组[（1．13±0．40）％]及对照shRNA组[（1．15±0．49）％]明显升高（P〈0．05）。而各组细胞晚期凋亡率无明显变化（P〉0．05）。实时定量PCR法证实shNANOG-1及shNANOG．2转染的细胞中TP53、PMAIPl、CASP9等基因表达较未转染组及转染非特异shRNA组显著升高（P〈0．05），而Bcl-2基因表达则明显下降（P〈0．05）。结论NANOG在多种人T-ALL细胞系中表达，下调NANOG的表达可引发T_ALL细胞凋亡。

Objective To explore gene expression of NANOG in T-cell acute lymphoblastic leukemia （T-ALL） cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells. Methods Real-time PCR （RT-PCR） and Westem blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells （Annexin V ＋/7-AAD-） and late apoptotic cells （Annexin V V7-AAD＋） were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected. Results Both NANOG gene and protein expression was positive in MOLT-4 and CCRF- HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-shcontrol were successfully constructed, as evidenced by the viral titers （ 1.83-3.12 ） × 10^8 IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences （shNANOG-1 and shNANOG-2） could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1 [ （8.06±1.61）%] and shNANOG-2 [（5.67±1.59）% ] were significantly increased as compared to that of MOLT-4 group [ （ 1.13±0.40）% ] or sh-control [ （1.15±0.49） % ] （P 〈 0.05 ）. However, no statistical difference among them was observed for late apoptotic cells （P〉0.05）. The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG- 2 group was augmented as compared to that of MOLT-4 group or sh-control （P〈0.05）. Reversely, a significant down-regulation of Bcl-2 gene expression was observed （P〈0.05）. Conclusion NANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.