The Function Of C3aR1Gene In Leukemia Cell Lines

Part one. Construction of Venus Vector Carrying C3aR1GeneObjective: Venus vector carrying the complement component3a receptor1gene wasconstructed to explore its function in acute myeloid leukemia cell lines.Methods:1.Human C3aR1gene was cloned and connected to the pMD19vectorbefore sequencing；2. The C3aR1gene was recombined with the transfer plasmid-Venusand transfected into293T cells by DNA-calucium phosphate. The virus particles werecollected and stored in-80℃refrigerator for further transfection.Results: The results showed that the sequence of cloned C3aR1gene cloned was thesame as that in GenBank．Which was further confirmed by the size of product digested byrestriction endonuclease BamHI.GFP expression was observed in293T and HL-60cellsunder the fluorescent microscope.Conclusion: Venus vector carrying C3aR1gene has been successfully constructed. Part two. The expression and function of C3aR1gene in leukemia celllinesObjective: To explored the effect of C3aR1gene and C3a-C3aR axis on leukemia celllines, HL-60and K562.Such experiments as proliferation, differentiation, apotosis,migration and cell cycle were performed.Methods: The transfect efficiency of lentivirus infection on HL-60cells wasperformed by measuring the expression of GFP on FCM after transfected for72hours. Theexpression level of C3aR1on HL-60and K562was detected with RT-PCR and FCM.Thecells were divided into three groups: cell with parental, Venus and C3aR1-Venus.Proliferation was determined by CCK-8and differentiation was examined by NBTreduction reaction. Cell cycles and apoptosis were analyzed by Flow cytometry.The trans-matrigel assay was employed to observate the reactivity of HL-60to the chemokinesSDF-1. Meanwhile, the function of C3a-C3aR axis was observed in these assays.Results: C3aR1didn’t affect the proliferation, differentiation, apoptosis and cell cycleon HL-60series which was further confirmed on K562cells.C3a-C3aR axis didn’t exertany effect on the proliferation, differentiation, cell cycle and apotosis by the experimentsof C3a stimulation at the concentration of1μg/ml. In the trans-Matrigel assay, C3ashowed its promoting effect on migration of HL-60cells transfected with C3aR1under thecontext of SDF-1(200ng/ml) compared with SDF-1alone. The OD value was1.45±0.07and2.27±0.17for chemotaxis by SDF-1alone and SDF-1with C3a(P=0.018)..Conclusion: C3aR1gene didn’t show its effect on proliferation, differentiation, cellcycle and apotosis, neither C3a-C3aR axis did. C3a-C3aR axis played its role in promotingHL-60reactivity to chemokine of SDF-1.. Part three. The effect of C3aR1on leukemia cell lines’ resistancechemotherapyObjective: To investigate the effects of C3aR1and C3a-C3aR1axis on thedrug-resistance to chemotherapy in HL-60and K562in vitro.Methods:1. Cytarabine, daunorubicin, etoposide, arsenic trioxide and ABT-737wereemployed to study the the effect of C3aR1on chemoresistance. HL-60and K562cellswere treated under different doses of the above chemo-drugs for24hrs and CCK8was usedto measure the proliferation inhibition on C3aR1-Venus group and control group.2. Theeffect of C3a-C3aR axis was observed on cells treated with VP-16and ABT-737by addingC3a at the dose of1μg/ml. Meanwhile, cell cycle was measure by FCM after the cellsexposed to VP-16for24hrs combining with C3a or not.Result: HL-60transfected with C3aR1-Venus showed a significantly resistance toetoposide and ABT-737compared with controls. OD values of HL-60transfected withVenus-C3aR, HL-60transfected with Venus control and parentals cells were0.28±0.027,0.49±0.022, and0.53±0.058,, respectively, after the cells were treated by VP-16under the concentration of100ng/ml (P=0.007). and0.12±0.021,0.29±0.026, and0.27±0.082, correspondingly, after the cells were treated by ABT-737at the dose of100nMfor24hrs (P=0.011).K562cells only showed resistance to VP16after transfected byVenus-C3aR.2. C3a didn’t show any effects on chemoresistance and cell cycle to VP-16.3. Vp-16caused cell cycle arrested at G2phase which was7.0±2.13and57.0±4.24forHL-60and HL-60treated by VP-16respectively,(P=0.018). Compared with control group,the G2phase of C3aR1-Venus decreased (57.0±4.24vs42.0±2.18,P=0.033). However,C3a-C3aR axis didn’t show effect on cell cycles after adding C3a at the dose of1μg/mlthe percentage of G2phase was42.0±2.18and46.5±4.49, respectively (P=0.246). Vp-16caused cell cycle arrested at G2phase which was40.4±2.19and61.9±2.41for K562andK562treated by VP-16respectively (P=0.011). Compared with control group, the G2phase of C3aR1-Venus decreased (61.9±2.41vs45.3±3.74,P=0.023). However,C3a-C3aR axis didn’t show effect on cell cycles after adding C3a at the dose of1μg/mlthe percentage of G2phase was35.3±5.23and41.5±4.94, respectively (P=0.204)。Conclusion:1. C3aR1expression could enhance HL-60cells’ resistance to VP-16and ABT-737and K562cells’ resistance to VP-16. VP-16could cause cell cyclearrestedvon G2phase,K562could cause cell cycle arrested on G2phase and C3aR1expression relieved this process.. Part four. The clinical significance of C3aR1in acute myeloid leukemiaObjective: In vitro,C3aR1demonstrated involving in migration and resistance toEtopos which indicated that it may play a role in acute leukemia(AL). In this section, weinvestigated C3aR1transcriptional level in acute myeloid leukemia(AML) at differentstage and its clinical significance in AML.Methods:319cases with acute myeloid leukemia(AML) patients at different stagesfrom the first affiliated hospital of Soochow University during2007to2011were enrolledin this study which include121cases at initial stage,163cases at complete remission(CR) and35cases at relapse. Transcriptional levels of C3aR1was detected in bone marrowsamples of the patient and20controls without leukemia using real-time quantitativeRT-PCR analysis(qRT-PCR).Results:1.In the cohort of319adult patients with AML, the transcriptional levels ofC3aR1in the patients at initial stage was significant high compared with that at CR. C3aR1level surged high after relapse. The transcriptional levels of C3aR1in de novo and relapseAML patients revealed no significant difference(P=0.242),but were both higher than that inCR(P﹤0.05).2.In28patients with AML-M2under the age of60years, high C3aR1transcriptional levels had shorter overall survival(OS) than those with low expression(P=0.043).3. In23patients with AML/ETO fusion gene positive, high C3aR1transcriptional levels also had shorter OS than those with low expression (P=0.045).Conclusion: Our results demonstrated that the transcriptional levels of C3aR1wassignificantly associated with the progression of AML, and high transcriptional levels ofC3aR1plays a role as a negative prognostic factor. Combined with our previousexperimental data in vitro, C3aR1may be a negative biomarker in AML via promotingmigration and resistance to chemotherapy.