title: Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells.
abstract: Nucleophosmin, a multifunctional nucleolar phosphoprotein, is involved in many cellular activities. However, the role of NPM in drug-resistance of leukemia has not yet been explored. We designed and selected one shRNA targeting on NPM gene transduction into HL-60 and HL-60/ADR cell lines (an adriamycin resistant cell line) by lentivirus. Cell proliferation, apoptosis and differentiation were assessed. The expressions of the related genes and proteins were detected by real-time quantitative RT-PCR and Western blotting. The results showed obvious down-regulation of NPM mRNA and protein levels after NPM RNAi. NPM-targeted RNAi also resulted in many cellular changes, such as, suppressing cell proliferation and inducing cell differentiation. Down-regulation of NPM gene could arrest the cell cycle progression, an increase in the proportion of G0/G1 phase in knockdown groups. NPM gene silencing could also induce pro-apoptotic genes and proteins expression, and inhibit anti-apoptotic genes/proteins expression. Furthermore, IC50 of two chemotherapeutic agents (adriamycin and ADR; daunorubicin and DNR) to HL-60 and HL-60/ADR cells decreased, especially more remarkable on HL-60/ADR cells. IC50 of ADR on HL-60/ADR cells was reduced from 12.544 ± 0.851 μmol/L (before NPM RNAi) to 6.331 ± 0.522 μmol/L (after NPM RNAi), IC50 of DNR was reduced from 2.152 ± 0.143 μmol/L (before NPM RNAi) to 1.116 ± 0.093 μmol/L (after NPM RNAi). The relative reversal rate of HL-60/ADR cells on ADR was 50.2%, and on DNR was 48.9%. In conclusion, our results demonstrated that shRNA expression vectors could effectively reduce NPM expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents.
authors.affiliation: Fujian Institute of Hematology, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou 350001, Fujian, China.
authors.forename: Minhui
authors.initials: M
authors.lastname: Lin
authors.forename: Jianda
authors.initials: J
authors.lastname: Hu
authors.forename: Tingbo
authors.initials: T
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Buyuan
authors.initials: B
authors.lastname: Chen
authors.forename: Xinji
authors.initials: X
authors.lastname: Chen
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D048708:Cell Growth Processes / Q000235:genetics; D004317:Doxorubicin / Q000627:therapeutic use; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D016086:Lentivirus; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D000090243:Nucleophosmin; D004317:Doxorubicin
keywords: 12-O-tetradecanoylphorbol-13-acetate; ADR; AML; DNR; ECL; IC50; LV; Leukemia; MDR; Multidrug resistance; NBT; NPM; Nucleophosmin; PI; RNA interference; RNAi; RR; TPA; VCR; VP16; acute myeloid leukemia; adriamycin; daunorubicin; enhanced chemiluminescence; etoposide; half inhibitory concentration; lentivirus; multidrug resistance; nitroblue tetrazolium reduction; nucleophosmin; propidium iodide; qRT-PCR; quantitative real-time reverse transcription chain reaction; relative reversal rate; shRNA; short hairpin RNA; vincristine