The Functionof CCL2 Gene In Leukemia Cell Lines

Part one. Construction of Lentiviral Vector Carrying CCL2GeneObjective: To explore the function of chemokine C-C motif ligand 2 gene(CCL2) in acute myeloid leukemia cell lines, CCL2 gene was constructed into Lentiviral vector.Methods:.Human CCL2 gene was cloned from U937 and connected to the p MD19 vector before sequencing.The CCL2 gene was recombined with the transfer plasmid-PLVX and transfected into 293 T cells by DNA-calucium phosphate. The virus particles were collected and transfected into THP-1 cells.Genomic DNA as a template, we used PCR to amplify the CCL2 gene.Results: The results showed that the sequence of cloned CCL2 gene cloned was the same as that in Gen Bank, which was further confirmed by the size of product digested by double restriction endonuclease. The overexpression of CCL2 was valideted on the level of DNA.Conclusion: Lentiviral vector carrying CCL2 gene has been successfully constructed. Part two. The function of CCL2 gene overexpressed in leukemia cell linesObjective: To explore the effect of CCL2 gene on leukemia cell line THP-1,following experiments such as proliferation, apotosis, cell cycle, migration and drug-resisitance were performed.Methods: The cells coming from parental, PLVX Vectors and CCL2-PLVX were used. Proliferation was determined by CCK-8. Cell cycles and apoptosis were analyzed by Flow cytometry. Daunorubicin and Homoharringtonine at different dose were performed on above cells for 24 hrs and CCK8 was used to measure the cell vability.The chemotaxis of THP-1 to CCL2 at a concentration of 100ng/ml was detected. Meanwhile, the transwell assay was run to compare the migration and invasion ability of three kinds of cells.Results: With the Trans-Matrigel assay, recombinant protein of CCL2 promoted THP-1 cells migration. High concentration of CCL2 was detected in the culture at the above cell of Trans-Matrigel which decreased the invasion abilityof CCL2 overexpressed cell lines(CCL2-PLVX cells).CCL2 didn’t affect the proliferation, apoptosis, cell cycle and chemosensitivity on THP-1.Conclusion: CCL2 gene plays a role in promoting migration and invasion of AML cell line. Part three. The effects of CCL2 RNA interferance on leukemia cell linesObjective: To explore the effect of CCL2 konckdown by RNA interferenceon on leukemia cell line HL-60, following experiments such as proliferation, apotosis, cell cycle were performed.Methods: Lentivirus with CCL2-knockdown was successfully constructed after screening effective CCL2 sh RNA sequence and transduced into HL-60 cells which was validated on the level of m RNA and protein by real-time PCR and Western blot. The cells coming from parental, sh-Vectors and sh CCL2 were detected for cell growth viability by CCK-8 assay, cell cycles and apoptosis by Flow cytometry.In order to analysis the different gene expression between the CCL2 knockdown and the control by c DNA expression profiling technology.Results: Low expression of CCL2 significantly decreased HL60 cell growth. The CCL2-sh RNA-transduced HL-60 cells showed 12% shift from S phase to G1 phase of the cell cycle. The results of expression profiling showed that there are total 159 differentially expressed genes related to cell cycle, NOD-like receptor signaling pathway, TNF signaling pathway and NF-kappa B signaling pathway, meanwhile, Cyclin D1 was decreased on the level of m RNA and protein.Conclusion:These data suggest that cyclin D1 mediated the effect of CCL2 on cell proliferation likely via downregulating more cells arrested at G1 phase. Part four. The clinical significance of CCL2/CCR2 in pediatric acute leukemiaObjective: To explore the relation of CCL2 and its receptor CCR2 with clinical features, the m RNA expression level of CCL2 and CCR2 was measure in pediatric acute leukemia.Methods: 176 cases with acute leukemia(AL) at different stages were detected for the expression of CCL2 and CCR2 by real-time RT-PCR(q RT-PCR), namely,50 pediatric patients of AML(initial = 32, CR = 18) and 126 patients of acute lymphoid leukemia(ALL)(initial = 66, CR = 60), AML-M3 was excluded.All the sample were collected during 2010 to 2012 from the Children’s Hospital of Soochow University. Twenty samples without leukemia were used as control.Results: Initial ALL patients had significantly lower transcript levels of CCL2 and CCR2 comparing with patients in CR(PCCL2 and PCCR2<0.001) and control group(PCCL2 and PCCR2<0.001). In AML patients, the expression level of CCL2 was lower than control(p<0.001), and stayed at a low expression even after chemotherapy. CCR2 didn’t show significant difference between AML patients and control(P>0.05). Interestingly, CCL2 and CCR2 expression in de novo AML were higher than in ALL(PCCL2= 0.002 and PCCR2<0.001). CCL2 expression level upon diagnosis didn’t show any relation with early treatment outcome in ALL and AML patients considering the responses of corticosteroid pretreatment and induction chemotherapy.Conclusion: Our results demonstrated that the transcriptional level of CCL2 was significantly lower in de novo acute leukemia.