Effect Of TBLR1-RARα On Erythroid Differentiation Of K562 Cells

Objective:In our previous study, a new t(3;17)(q26;q21) chromosomal translocation in an intractable case was observed. A novel RARa fusion gene TBLR1-RARα, which has not been reported, was identified by 5’rapid-amplification of cDNA ends (5’RACE) technique. The purpose of this study is to explore the effects of TBLR1-RARα on erythroid differentiation induction of leukemia cell line K562 cells, and to investigate its related mechanisms.Methods:The tetracycline (Tet) inducible Tet-Off system was used to construct the TBLRl-RARa expression plasmid pLVX-Tight-Puro-TBLR1-RARα-flag, and then transfected into 293T cell line by PEI. K562 cell line was infected, and TBLR1-RARα expressed K562 (K562-TR) was screened by G418 and puromycin.Based on the Tet-Off inducible system, the expression of TBLR1-RARα was shutted off when K562-TR cells were exposed to doxycycline (Dox). The real-time quantitative RT-PCR (qTR-PCR) was performed to detect the erythrocyte related gene expreesion of CD71,α, ε and γ-globins after K562-TR cells were induced to differentiation by all-trans retinoic acid (ATRA). The flow cytometry analysis was used to determine the expression of surface markers CD71 and CD235a, and the benzidine staining to analyze hemoglobin synthesis in ATRA treated K562-TR cells.Results:The results showed that the gene expression level of CD71, α,ε,γ-globins of K562-TR cells increased when TBLR1-RARα was expressed.The proportion of CD71+ cells detected by the flow cytometry also increased. Benzidine staining showed that ATRA treatment could induce hemoglobin synthesis in TBLR1-RARα fusion gene expressed K562-TR cells.Conclusions:The expression of TBLR1-RARα fusion gene could induce the erythroid differentiation of K562 cells.