Expressions And Mechanism Of Beta-arrestins In Leukemia

Objective: To investigate the expression and significance ofβ-arrestin1 andβ-arrestin2 in leukemia patients.Methods: Ninety-five newly diagnosed leukemia patients (Leu group) were enrolled, which were divided into acute myeloiblastic leukemia (AML) subgroup (30 cases), acute lymphoblastic leukemia (ALL) subgroup (44 cases) and chronic myeloid leukemia (CML) subgroup (21 cases) according to different leukemia subtypes. Thirty-six sex- and age-matched patients without malignant hematological diseases were choosed in this study as the control (NL group). The bone marrow and periphery blood samples were simultaneously collected, then mononuclear cells of these samples were isolated, and the mRNA expressions ofβ-arrestin1 andβ-arrestin2 were detected by quantitative real-time RT-PCR, and the protein expressions were detected by Western blot and immunofluorescence assays, respecitvely.Results: All the mRNA and protein expressions ofβ-arrestin1 andβ-arrestin2 were detected in the mononuclear cells from both bone marrow and periphery blood of Leu group and NL group, and the expression levels were significantly higher in Leu group than in NL group (P<0.01). Compared with NL group, both the mRNA and protein expressions ofβ-arrestin1 andβ-arrestin2 in AML, ALL and CML subgroups were all up-regulated(P<0.05 or P<0.01), but there were no statistical differences ofβ-arrestin expressions among these three subgroups(P>0.05).Conclusion: The expressions ofβ-arrestin1 andβ-arrestin2 are significantly up-regulated in bone marrow and periphery blood mononuclear cells of leukemia patients, indicating thatβ-arrestin1 andβ-arrestin2 might be related to the pathogenesis and progression of leukemia.PartⅡPackaging ofβ-arrestins recombinant lentivirus and screening of K562 stable cell linesObjective: To pack recombinant lentivirus vectors carryingβ-arrestin1/2 gene or siRNA fragment ofβ-arrestin1/2, and to obtain the stable leukemia K562 cells by infecting with these recombinant lentivirus and screening.Methods: The three-plasmid co-transfection system was applied to package the recombinant lentivirus ofβ-arrestins in 293T cells. The titers of the lentivirus were tested through infected 293T cells with diluted lentivirus. After that, K562 cells were infected with thoseβ-arrestins recombinant lentivirus. The infect efficiency was evaluated by real-time quantitative PCR and Western blot. K562 stable cells expressingβ-arrestins were constructed by limiting dilution analysis.Results: GFP expressions were observed by fluorescence microscope after co-transected three lentivirus package plasmids into 293T cells 48h later. The titers of these lentivirus were as follows: LV-EV, 2.21×10~7 pfu/ml; LV-βArr1, 1.17×10~7 pfu/ml; LV-siβArr1, 1.96×10~7 pfu/ml; LV-βArr2, 1.37×10~7 pfu/ml; LV-siβArr2, 1.64×10~7 pfu/ml. After infection K562 cells, both the mRNA and protein expressions ofβ-arrestin1 andβ-arrestin2 in LV-EV group were no significant alterations, while theβ-arrestin1 mRNA and protein expressions were up-regulated in LV-βArr1 group but down-regulated in LV-siβArr1 group individually.β-arrestin2 mRNA and protein expressions were up-regulated in LV-βArr2 group but down-regulated in LV-siβArr2 group, respectively.Conclusion: The recombinant lentivirus of LV-βArr1, LV-siβArr1, LV-βArr2, LV-siβArr2 and LV-EV are successfully packaged. These recombined lentivirus could effectively infect K562 cells and induce the correspondingly expression alterations ofβ-arrestin1 andβ-arrestin2. Objective: To observe the effects ofβ-arrestin1 recombinant lentivirus on K562 cell proliferation, and to preliminarily explore its potential mechanism.Methods: The mRNA expressions ofβ-arrestin1 in K562-EV, K562-βArr1 and K562-siβArr1 cells were detected by real-time quantitative RT-PCR. The cell proliferation was measured by MTT analysis. The gene expressions of MMP-2, MMP-9, bax and bcl-2 were detected by real-time quantitative RT-PCR.Results: Both the mRNA and protein levels ofβ-arrestin1 were significantly elevated in K562-βArr1 cells and decreased in K562-siβArr1 cells (P<0.01). Compare to normal K562 cells and LV-EV K562cells, the proliferation degrees were significantly elevated in K562-βArr1 cells, but were significantly decreased in K562-siβArr1 cells (P<0.01). The mRNA expression of MMP-9 was up-regulated in K562-βArr1 cells, down-regulated in K562-siβArr1 cells compared with K562-EV cells (P<0.05). There were no significant differences between the K562-EV and control. However, there were no significant alterations of MMP-2, bax and bcl-2 mRNA expression among these groups.Conclusion:β-arrestin1 could promote cell proliferation in K562 cells, which might associate with the enhanced MMP-9 mRNA expression to alter the microenvironment.