Construction and Identification of Leukemia Cell Line Stably Expressing CD123 and CLL-1 / 中国实验血液学杂志

OBJECTIVE@#To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.@*METHODS@#The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.@*RESULTS@#The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10@*CONCLUSION@#Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.

Effects of sodium arsenite and arsenic metabolites on the expression of linear and circular RNA of Nup107 in A549 cells / 中国职业医学

OBJECTIVE: To explore the effect of sodium arsenite and arsenic metabolites monomethylarsenic acid(MMA) and dimethylarsenic acid(DMA)on the expression of linear and circularRNAs of nucleoporin 107(Nup107) in human lung adenocarcinoma A549 cells. METHODS: i) The A549 cells in logarithmic phase were treated with 0, 30, 60, 90 μmol/L sodium arsenite for 48 hours. ii)The A549 cells in logarithmic phase were treated with 90 μmol/L sodium arsenite, MMA and DMA for 48 hours, the control group received no treatment. After culturing, the relative expression of linear RNA and circular RNA(circRNA) of Nup107 was detected by real-time quantitative polymerase chain reaction. RESULTS: i) The relative expression of linear RNA of Nup107 decreased and four circRNA isomers such as hsa＿circ＿0003599, hsa＿circ＿0027477, hsa＿circ＿0027478 and hsa＿circ＿0027479 increased with the increase of sodium arsenite dose(all P<0.01), showing a dose-effect relationship. In the 90 μmol/L sodium arsenite stimulated group, the relative expression of linear RNA of Lin-Nup107 decreased, and the four circRNA isomers increased compared with the control group in the A549 cells(all P<0.01). ii) The relative expression of Lin-Nup107 increased in the MMA and DMA stimulated groups and decreased in the sodium arsenite stimulated group compared with the control group in the A549 cells(all P<0.05). The relative expression of Lin-Nup107 decreased in the sodium arsenite stimulated group compared with the MMA and DMA stimulated groups in the A549 cells(all P<0.05). The relative expressions of hsa＿circ＿0003599, hsa＿circ＿0027478, hsa＿circ＿0027479 in the sodium arsenite stimulated group were higher than that in the MMA and DMA stimulated groups as well as the control group(all P<0.05).The relative expressions of hsa＿circ＿0027479 in the DMA stimulated group was lower than that in the control group(P<0.05). CONCLUSION: Sodium arsenite can induce the down-regulation of linear RNA and the up-regulation of circRNA of Nup107 in a dose-dependent manner. The metabolites of arsenic MMA and DMA can induce the overexpression of linear RNA of Nup107, however, they had no obvious effect on the circRNA of Nup107 in A549 cells.