title: [Effects of NANOG gene down-regulation on the apoptosis of T-cell acute lymphoblastic leukemia cells].
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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Cao
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Lyu
authors.forename: Fan-jing
authors.initials: FJ
authors.lastname: Meng
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.forename: Chong
authors.initials: C
authors.lastname: Chen
authors.forename: Ling-yu
authors.initials: LY
authors.lastname: Zeng
authors.forename: Zhen-yu
authors.initials: ZY
authors.lastname: Li
authors.forename: Kai-lin
authors.initials: KL
authors.lastname: Xu
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D015870:Gene Expression; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D000071317:Nanog Homeobox Protein; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D034741:RNA, Small Interfering