The Mechanism Of IL-1α Propiece Regulation

Part I: Construction of an IL-1α Propiece Expression SystemObjective: The N-terminus of the IL-1α protein contains a nuclear localization sequence, which mediates nucleus translocation of IL-1a. However, the specific function of the nuclear IL-1α is not clear. Previous studies have shown that the level of IL-1 in leukemic cells was abnormally high. To investigate the biological function and possible cellular regulatory mechanism of the nuclear IL-1α propiece in leukemic cells, we constructed an eukaryotic expression system for the IL-1α propiece.Methods: IL-1 α propiece gene was cloned into a lenti-virus vector and infected Jurkat cells. Cells with high expression of IL-1α propiece were sorted by flow cytometry. The cellular localization of IL-1α propiece was analyzed by confocal microscopy using flag tag protein. We also isolated nuclei from the trasnfected cells and detected the existence of the protein in the nuclei by flowcytometry using an anti-flag antibody. Additionally, we extracted nuclear protein and cytoplasmic protein and examined the presence of IL-1 propiece by Western blotting analysis to detect the expression of IL-1α propiece in these two components.Results: The IL-1α propiece gene was successfully cloned into the lenti-virus expression vector, and the virus particles were packaged and infected into Jurkat cells. The infected cells were sorted based on GFP expression. The flag tag protein and DAPI represent IL-1a and nucleus respectively during confocal microscopic detection. The confocal study revealed that IL-1a and DAPI localized in the same location. Furthermore, our flowcytometric analysis showed that the expression of IL-1α propiece is in the nucleus and our Western blotting analysis also demonstrated that the expression of IL-1α propiece is in the nuclear compartment. These results clearly proved that the IL-1α propiece is indeed located in the nucleus.Conclusion: An IL-1α propiece eukaryotic expression system was successfully constructed. Jurkat cells with high expression of the IL-1α propiece were successfully established. Our results from confocal microscoopy, flowcytometric analysis and western blotting analysis showed that the IL-1α propiece is located in the nucleus.Part II: The Biological Effercts of IL-1α propiece in Jurkat CellsObjective: The experiments were designed to investigate the effects of nuclear IL-1α propiece on cell proliferation, cell cycle progression and apoptosis in acute lymphacytic leukemic cells. We also examined the biological effects of IL-1α propiece expression in acute lymphacytic leukemic cells under low serum stress and upon treatment with anticancer drugs.Methods: MTT assay was used to detect the proliferation of cells at 24, 48 and 72 hours in IL-1α propiece expressing acute lymphocytic leukemic cells. Cell proliferation under low serum stress and cytotoxic treatment with cisplatin was also evaluated. The effect of IL-1α propiece on apoptosis in leukemic cells was analyzed by Annexin V and 7AAD double staining. PI staining was used to detect the effect of IL-1α propiece on cell cycle progression of leukemic cells under low serum stress and treatment with cisplatin.Results: IL-1 α propiece overexpression can promote the proliferation of Jurkat cells and P388 cells, increase the percentage of G2/M cells, and reduce the level of apoptosis upon treatment with cisplatin and exposure to low serum stress.Conclusion: IL-1α propiece overexpression can promote the proliferation of acute lymphacytic leukemic cells, inhibit their apoptosis and increase the percentage of G2/M phase cells.Part III Detection and data mining of microarray data from IL-1α propiece overexpression systemObjective: To evaluate the effect of IL-1 propiece overexpression on gene expression.Methods: Changes in gene expression profile induced by IL-1α propiece overexpression was determined by the Agilent Human 4 x 44 K gene expression array. Cluster analysis of the expression resulted in distinct groups based on the expression of MT gene and NF-κB targeted genes. Enrichment analysis from Gene Go and GO(Gene Ontology)database. IL-1α propiece 3D structure was predicted on the I-TASSER server. The DNA binding motifs were predicted on DBD-Hunter, Bind N, DISPLAR and i DBPs server. The co-regulated promoter was enrichment by PRIMA.Results: Hierarchical clustering revealed that the MT gene family and NF-κB targeted genes were upregulated in IL-1α propiece overexpressing cells. There are about 110 cellular and molecular processes in which contents are defined and annotated by Gene Go. The angiogenesis and anti-apoptosis pathways were implied to be affected by IL-1α propiece. IL-1α propiece was predicted to have DNA binding ability and the Sp1 promoter was predicted to be involved by the PRIMA enrichment assay.Conclusion: IL-1α propiece exerts its biological functions through regulating multiple dynamic signal transduction networks.Part Validation of the Pathways Affected by ILⅣ-1α Propiece Expression and Screening for Protein-DNA Interaction MotifsObjective: To validate the signalling networks affected by IL-1α propiece expression.To screen for protein-DNA interaction motifs in the Sp1 promoter.Methods: Validate specific ling pathways affected by IL-1α propiece expression using Westen blotting analysis. Construct Sp1 promoter mini library. IL-1α propiece gene was also cloned into p GEX-6p prokaryotic expression vector. The expression of IL-1α recombinant protein was induced by IPTG and purified by GST tag protein. The recombinant IL-1α propiece was assayed in the SELEX with Sp1 promoter mini library.Results: IL-1α propiece upregulated IKK and decreased Ik B Expression, which in turn resulted in P65 translocation to the nucleus. The phosphorylation of AKT was upregulated by IL-1α propiece overexpression. The p38 MAPK phosphorylation was increased. The levels of Caspase 8, caspase3, PARP were decreased and Bcl-2 and HSP-70 were upregulated by IL-1α propiece overexpression. The high affinity binding site was approved by IL-1α propiece SELEX with Sp1 promoter mini library.Conclusion: NF-κB,AKT and p38 MAPK can be activated by IL-1α propiece overexpression. Apoptosis gene was downregulated and anti-apoptosis gene was upregulated. DNA fragments screened by SELEX have a CG rich pattern that could predict a sequence logo.