[Expression of NANOG gene in acute lymphoblastic leukemia cells and construction of lentiviral vector carrying NANOG specific shRNA].
The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was detected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT-4 cells with the lentivirus constructs, GFP (+) cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were constructed. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were (1.83-3.12) ×10(8) IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP(+) cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could significantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulate the expression of NANOG mRNA are obtained.
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Jiang
J
Cao
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Fan-Jing
FJ
Meng
Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Li
L
Li
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Chao
C
Lu
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Jun
J
Zhou
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Hai
H
Cheng
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Wei
W
Chen
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Chong
C
Chen
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China. E-mail: lihmd@163.com.
Kai-Lin
KL
Xu
D000328:Adult; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D005260:Female; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D000071317:Nanog Homeobox Protein; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D055815:Young Adult
D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D012333:RNA, Messenger; D034741:RNA, Small Interfering