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Effects of Downregulating TSG101 on the Phenotype of Human Leukemia Cells
Li-Hua Liu*, Hong-Wei Zhang1, Zhong-Xiang Mi2
Laboratory of Molecular Diagnosis, Medical College, Shaoxing University, Shaoxing 312000, China; 1Department of Neurology, Affiliated Hospital of Medical College, Shaoxing University, Shaoxing 312000, China;2College of Life Sciences,
Abstract: The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination， then transfected into HL-60 cells. Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blot analysis. The growth curve was made using MTT assay. Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferous indexes were calculated. The apoptosis after CDDP treatment was detected by DNA ladder and annexin V/propidium iodide binding analyses. The expressions of P-gp and MRP were analyzed by Western blot. mU6pro-TSG101 siRNA was successfully constructed and transfected into HL-60 cells. Down-regulation of TSG101 could significantly suppress the proliferation of HL-60 cells with a G1 cell cycle arrest， and enhance the sensitivity of HL-60 cells towards CDDP-induced apoptosis. The expressions of P-gp in transfected cells was decreased as compared with that of the control， while MRP not. Therefore， down-regulation of TSG101 could suppress the proliferation of HL-60 cells， and enhance the sensitivity of HL-60 cells to conventional chemotherapeutic agents to a degree， suggesting TSG101 could be used for gene therapy in future.
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