title: [Establishment of nucleophosmin gene silenced HL-60 and its resistant cell line].
abstract: This study was aimed to construct model cell line of NPM1-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E.coli DH5α. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2.0 and pGCSIL-GFP-NPM1-shRNA were cotransformed into 293T cells by lentivirus vector system. NPM1-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic expression vector pGCSIL-GFP-NPM1-shRNA was constructed. pGCSIL-GFP-NPM1-shRNA was packed into NPM1-RNAi-LV by lentivirus vector system, and transfected into HL-60 and HL-60/ADR cell lines. At mRNA level, the efficiency of NPM1 mRNA knockdown was more than 90% (p < 0.05). At protein level, obvious down-regulation of NPM protein was noted, indicating that NPM1 gene in HL-60 and HL-60/ADR cell lines was knocked down after transfected with NPM1-RNAi-LV. The resistance of HL-60/ADR cell line to adriamycin decreased to a certain degree after NPM1 gene silencing. It is concluded that the model cell lines of NPM1-RNAi in HL-60 and HL-60/ADR are successfully constructed, which can be used for investigating the potential role of NPM1 gene in drug resistance of leukemia.
authors.affiliation: Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fujian Province, China.
authors.forename: Min-Hui
authors.initials: MH
authors.lastname: Lin
authors.forename: Jian-Da
authors.initials: JD
authors.lastname: Hu
mesh_terms: D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D034622:RNA Interference
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D004317:Doxorubicin