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Zhonghua Xue Ye Xue Za Zhi. 2013 Dec;34(12):1001-5. doi: 10.3760/cma.j.issn.0253-2727.2013.12.001.

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

[Article in Chinese]

Author information

  • 1Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.

Abstract

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 Western 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⁺/7-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-sh control were successfully constructed, as evidenced by the viral titers (1.83-3.12)× 10⁸ 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.

PMID:
24369153
[PubMed - indexed for MEDLINE]
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