Human Hyal1 Gene Transfection

PART ONE Construction of Eukaryotic Expression Vector of Human HYAL1 Gene and Its Expression in MCF-7 and ZR-75-30 CellsObjective Construct the eukaryotic expression vector of human HYAL1 gene and obtain MCF-7 and ZR-75-30 cell clones expressing HYAL1 gene stably and established base for the further research on the relationship between HYAL1 gene and the invasion and metastasis of Breast cancer .Methods the cDNA encoding the HYAL1 gene of human breast cancer was amplified by RT-PCR from the total RNA isolated from the human MDA-MB-435S cells and was inserted into pcDNA3.1/V5-His-TOPO vector to construct the recombinant eukaryotic expression vector .The recombinant plasmid was then transferred into MCF-7 and ZR-75-30 cells .Results Using RT-PCR,a 1332bp DNA fragment was successfully amplified from the human MDA-MB-435S cell, Restriction enzyme digestion analysis and DNA sequencing showed that the HYAL1 gene was inserted into recombinant vector. RT-PCR analysis revealed that HYAL1 gene could be expressed stably in the transfected MCF-7 and ZR-75-30Conclusion The eukaryotic expression vector of human HYAL1 gene was successfully constructed and the MCF-7 and ZR-75-30 cell clones that can express the HYAL1 gene were obtained PART TWO Effects of HYAL1 gene overexpression on invasive ability of breast cancer cellsObjective To study the effects of HYAL1 gene overexpression on invasive ability of breast cancer cell lines MCF-7 and ZR-75-30.Methods Double-chamber co-culture technique was applied to construct the invasive model in vitro, which was used to detect the invasive potential of breast cancer cell.Results Breast cancer cells overexpressing HYAL1 gene showed stronger invasive potential than control cells.Conclusion Overexpression of HYAL1 gene can promote the invasion of breast cancer cells in vitro. PART Three Effects of HYAL1 gene overexpression on angiogenic and ability of breast cancer cellsObjective To study the effects of HYAL1 gene overexpression on angiogenic ability of breast cancer cell lines MCF-7 and ZR-75-30.Methods Double-chamber co-culture technique was applied to construct the angiogenic model in vitro, which was used to detect the angiogenic potential of breast cancer cell.Results Breast cancer cells overexpressing HYAL1 gene showed stronger angiogenic potential than control cells.Conclusion Overexpression of HYAL1 gene can promote the angiogenesis of breast cancer cells in vitro. PART Four Effects of HYAL1 gene overexpression on proliferative ability of breast cancer cellsObjective To study the effects of HYAL1 gene overexpression on proliferative ability of breast cancer cell lines MCF-7 and ZR-75-30.Methods MTT and flow cytometry was used to detected the proliferation of breast cancer cells.Results Breast cancer cells overexpressing HYAL1 gene have no significant differrence on proliferative potential.Conclusion Overexpression of HYAL1 gene have no significant effects on proliferation.