Influence Of Aiolos Overexpression On Cell Biological Behaviours And Chemosensitivity In Leukemic Cells

Objective:1. To detect the expression level of Aiolos in four leukemic cell lines and normal child peripheral lymphocytes.2. To construct a recombinant lentivirus vector with enhanced green fluorescent protein, and transfer the vector into Jurkat cells to measure transfection efficiency.3. To explore the influence of Aiolos gene over-expression on Jurkat cells cycle, apoptosis, and chemosensitivity to etoposide.4. To insight the potential mechanism by measuring related genes involved in leukemic cells cycle and cell apoptosis regulation.Methods:1. Cells in logarithmic phase of four leukemic cell lines and normal child peripheral lymphocytes were collected, and Aiolos expression level of was detected by western blot.2. A recombinant lentivirus vector with Aiolos gene and enhanced green fluorescent protein was constructed and transfected into Jurkat cells. An Aiolos insert was isolated by polymerase chain reaction (PCR) amplification with two pairs of restriction primers. PCR products containing the entire Aiolos coding sequence were connected to PWPURO plasmid. The insert was cloned into the plasmid and was co-transfected into293T cells with packaging plasmids. The viral titer of the recombinant lentivirus vector PWPURO-Aiolos was detected with hole-by-dilution assay. PWPURO-Aiolos was transfected into293T cells indifferent Multiplicity of Infection (MOI), and detected green fluorescence protein expression4days later to ensure the efficient transfection and the optimum MOI.3. The virus vector was transfected into Jurkat cells, and the Jurkat cells were divided into three groups:Aiolos overexpression group (cells transfected with PWPURO-Aiolos), GFP transfected group (cells transfected with GFP plasmid) and non-transfection group (cells without transfected). After7days, Jurkat cells of the three groups were collected to detect cell cycle and apoptosis with MuseTM cell analyzer. The change of chemosensitivity to etoposide was observed with CCK8colorimeter. Related genes expression level was investigated with qRT-PCR.Results:1. Western blot analysis showed Aiolos expressed in Jurkat, K562, Molt-4and Nalm-6four different leukemic cell lines and child peripheral blood lymphocytes. The expression of Aiolos was significantly higher in T-lineage ALL cell lines Jurkat cells than in the B-lineage ALL cell line Nalm-6cells and T-lineage ALL cell line Molt-4cells, and was in lowest expression level in chronic myelogenous leukemia (CML) cell line K562cells among all cell lines. However, Aiolos expression in normal child peripheral blood lymphocytes was higher than it in the four leukemic cell lines.2. Human Aiolos gene over-expression vector (PWPURO-Aiolos) containing the entire Aiolos coding sequence was constructed successfully. Titer detection was up to3.11xlO8TU/ml measured with hole-by-dilution assay. An efficient transduction was ensured by visualizing enhanced green fluorescent protein (GFP; included in the vector) after4days transfection and the peak expression level was observed during4days to7days. Fluorescent microscope showed more than80%of the Jurkat cells were GFP positive in Aiolos overexpression group and GFP transfected group4days after transfection. Aiolos expression was significantly higher in the Aiolos over-expression group cells compared to GFP transfected group and non-transfection group(P<0.05). No compacious difference was found between GFP transfected group and non-transfection group (P>0.05)3. Cell cycle progression of Jurkat cells transfected with PWPURO-Aiolos was significantly arrested in G0/G1phase7days after transfection. Videlicet, The percentage of Jurkat cells in the G0/G1phase was much higher in Aiolos overexpression group cells than in GFP transfected group cells and non-transfection group cells (P<0.05).4. Cell apoptosis of Aiolos overexpression group cells was increased compared to GFP transfected group cells and non-transfection group cells (P<0.05). There was also a significant difference in both early apoptosis (P<0.05) and late apoptosis/dead (P<0.05) in Aiolos overexpression group cells compared to control groups cells. No compacious difference was found between GFP transfected group cells and non-transfection group cells.5. Cyclin D3, one of the known cell cycle-related genes, was down-regulated in Aiolos overexpression group cells. Furthermore, Skp2(a typical representative of the negative regulators to cell cycle) and Bcl-2(an anti-apoptotic gene) were also down-regulated in Aiolos overexpression group cells (P<0.05). In addition, P21and P27who are family members of cyclin-dependent kinase inhibitor (CKI) were also detected up-regulated in Aiolos overexpression group cells (P<0.05). No distinct changes were detected on Bax (a pro-apoptotic gene). However, there was an increase in the Bax to Bcl-2ratio.6. When treated with etoposide, cells of the three groups were killed in dose-and time-dependent manner. The cell survival rates were lower in Aiolos overexpressed group compared to control groups after36h and48h administration when exposed to40μg/mL etoposide (P<0.05). Conclusions:A lentivirus vector PWPURO-Aiolos is contructed successfully and transfected into Jurkat cells efficiently. Recombinant lentiviral vectors with Aiolos gene overexpression can inhibit cell cycle, accelerate cell apoptosis and strengthen chemosensitivity to etoposide of Jurkat cells.