Study Of The Na～+,K～+-ATPase β₁-subunit In Human Erythroleukemia K562 Cells Differentiation And Proliferation

OBJECTIVES:Na⁺,K⁺-ATPase is an transmembrane protein found in the eukaryote cells and is a key enzyme of the energy conversion system. It is responsible for translocating sodium ions, potassium ions, glucose, amino acids, and other nutrients across the cell membrane utilizing ATP as the driving force. This transport regulates the banlance and maintains the normal gradients of Na⁺ and K⁺ in mostly eukaryocytes, to keep the corpuscular volume, PH and the stabilization of internal environment, et al. Na⁺,K⁺-ATPase is composed ofα,βandγsubunit. In recent years, it has been suggested thatβ-subunit plays essential roles in the correct translation rate, synthesis of theαsubunit, and the function of Na⁺, K⁺-ATPase holoenzyme. As well as, theβ-subunit is also involved in cell adhesion, cell mobility. Recent studies have demonstrated the Na⁺, K⁺-ATPase activity and the Na⁺,K⁺-ATPaseβ₁-subunit (ATP1B1) mRNA expression were significantly lower in several cancer patients as compared to normal subjects. There is a tendency that reports about the relationship between ATP1B1 with the tumorous genesis, developmet,prognosis and therapy have being increased. In the present study,we detect the ATP1B1 mRNA expression and the change of the Na⁺, K⁺-ATPase activity during the differentiation of human erythroleukemia K562 cells.Furthermore, we constructed the ATP1B1 eukaryotic expression vector and ATP1B1siRNA, then transfected K562 cells, to study the effect of ATP1B1 during the differentiation and proliferation of K562 cells. We hope to offer evidences of ATP1B1 in tumor reseach.METHODS:1 The model which K562 cells were differentiated by DMSO was verified availability by NBT hydrogenization experiment and MTT method. Then we detected the ATP1B1 mRNA expression by real-time PCR and the change ofthe Na⁺,K⁺-ATPase activity during the differentiation.2 We used the gene recombination technology, inserted human ATP1B1 cDNA into pEGFP-C3 vector, to construct the ATP1B1 eukaryotic expression plasmid, and constructed ATP1B1siRNA by chemical, synthesis, then they were transfected into K562 cells by lipfectamin. While, ATP1B1 mRNA expression of the transformant was detected by RT-PCR, and Na⁺,K⁺-ATPase activity was detected after transfection, as well as, the cell morphology was observed by microscope, also, the proliferation of such cells was mearsured by MTT method.RESULTS:1 We verified the differentiation model of K562 cells which was established by 1.25%DMSO was valid by NBT hydrogenization experiment and MTT method, then detected the ATP1B1 mRNA expression increased gradually, and failed-off after the NO. 3 day. But the mRNA expression was always higher than that of the control. While, the Na⁺,K⁺-ATPase activity opposited to the ATP1B1 mRNA expression. The difference of every group was significant (P＜0.05).2 Inserteing human ATP1B1 cDNA into pEGFP-C3 vector used the gene recombination technology, we constructed the ATP1B1 eukaryotie expression plasmid. The recombined plasmid was digested with EcoR I, BamH I, then a 4kb and a 760bp fragment were shown after electrophoresis. This result is consistent with sizes of calculated, the direction and the base sequence of the plasmid were further verified by sequencing. The successfully constructed green fluorescent protein eukaryotic co-expressing vector was named pEGFP-ATP1B1. Then the plasmid was transfected into K562 cells by lipfectamin. After introduction of ATP1B1, the expression of ATP1B1 mRNA and the Na⁺,K⁺-ATPase activity were higher than others. While the cells were pyknosis, and the cells membrane were incomplete, as well as, the growth of the K562 cells transfected with ATP1B1 was inhibited obviously compared with the control group (P＜0.01), and the effect was more conspicuous after increasing dose of DNA. The difference of every group was significant (P＜0.05).3 The company constructed ATP1B1siRNA by chemical synthesis, and we checked the optimal interference site of ATP1B1 was NO. 950. Then ATP1B1siRNA was transfected into K562 cells by lipfectamin. After interference of ATP1B1, the expression of ATP1B1 mRNA was obviously lower than others detected by RT-PCR, but the changes both of the Na⁺,K⁺-ATPase activity and of the K562 cells proliferation were little (P＞0.05), the morphologicchange of cells were not obviously.CONCLUSIONS:1 The survey of the ATP1B1 mRNA expression and the Na⁺,K⁺-ATPase activity during the differentiation of K562 cells verified that ATP1B1 involved in tumor cell differentiation.2 The exogenous ATP1B1 transfeeted into K562 cells can increase both the ATP1B1 mRNA expression and the Na⁺,K⁺-ATPase activity, but inhibite the growth of the cells. It was suggested thatβ-subunit plays an important role in the growth of tumor cell.3 The ATP1B1siRNA constructed by chemical synthesis can interfered the expression of ATP1B1 mRNA, but the interferential effects both of the Na⁺,K⁺-ATPase activity and of the K562 cells proliferation were little.In a word, our finding displayed that ATP1B1 was relationship with the proliferation and differentiation of tumor cell, and ATP1B1 gene is promising to become a new target for anti-tumor therapy.