Eukaryotic Expression Plasmid Construction Of REGgamma Gene And Function Study In Human Breast Cancer Cell Lines

PART ONEEUKARYOTIC EXPRESSION PLASMID CONSTRUCTION OF REGgamma GENE AND CONSTRUCTION OF THE STABLE TRANSFECTIVE CELL LINEObjective.To construct the stable transfective cell line with the eukaryotic expression vector for human REGγ,cDNA to laid the foundation for further study of the function of REGγ.Methods:REGγcDNA was obtained by RT-PCR of total RNA extracted from the human breast cancer cell MCF-7,and it was digested by the restriction endonuclease EcoRI and EcoRV before connection with pcDNA3.1.The recombinant was transfected into HBL-100,MCF-7 and MDA-MB-231 cell by using lipofectamine2000.The stable transfected cell line was selected in medium containing antibiotic(G418).The expression of REGγ,detcected by immunocytochemistry,Western Blot,immunofluoresc ence and RT-PCR.Results:The recombinant was digested by restriction endonuclease and confirmed correct by sequencing,overexpression of REGγin the stable transfected cell line by contrast with untransfective cell line(P＜0.01);The expression of REGγ,in breast cancer cell lines were obviously higher than those of breast epithelial cell(P＜0.05);REGγlevels of MDA-MB-231 were obviously higher than that of MCF-7(P＜0.05).Conclusions.The construction of the eukaryotic expression vector for REGγwas successful,and the stable transfective cell line for overexpression REGγwas obtained by G418 selection,simultaneously, REGγexpressed in breast cancer cell lines were higher than those of breast epithelial cell;MDA-MB-231 was higher than that of MCF-7 within breast cancer cell lines.This work may pave the way for further study of the functions of REGγ.PART TWOFUNCTION STUDY OF REGγIN HUMAN BREAST CANCER CELL LINESObjective:To study the function of REGγgene in cell proliferation, apoptosis,and the effect for oncogene in human breast cancer cells.Methods:The effect of REGγgene expression on cell proliferation and cell cycle was examined by soft agar conoly formation test,MTT assay and flow cytometry(FCM);Immunocytochemistry was performed to detect the differences in proliferative cell nuclear antigen(PCNA),bcl-2,fas and c-myc in breast cancer cells;Apoptosis was measured via FCM,and caspase 3 expression was determined using spectrophotometry; Ultrastructural changes in breast cancer cells were observed with transmission electron microscopy(TEM).The effect of REGγgene on target protein P21,SRC-3 and CyclinD1 was detected by Western Blot.Results:MTT assay and FCM indicated that Cell growth was accelerated,the doubling time was shortened and the proportion of cells in proliferative phase in tansfected with REGγgroup was obviously higher than that of untransfected with REGγ.Soft agar conoly formation test indicated that the cell colony forming efficiency was increased,the time of colony forming was shortened and survival time of colony was long in tansfected with REGγ,in contrast to untransfected with REG_γ.The level of PCNA measured by immunocytochemistry indicated that the expression of PCNA was remarkably higher in cells transfected with the REGγ,gene (P＜0.01).The optical density(OD) was obviously lower in cells transfected with REGγby using spectrophotometry of caspase 3(P＜0.05), and the rate of apoptosis was lower in cells transfected with REGγ(P＜0.01).The expression of bcl-2 and c-myc were enhanced in cells transfected with recombinant,on the contrary,the expression of fas was decreased(p＜0.05).Karyosome hypertrophy,mitochondrion abundance, Golgi apparatus abundance and ectasis were seen with TEM.Apoptotic bodies were not observed.Western blot revealed that the expression of p21 and SRC-3 were highly decreased in cells stably transfected with the recombinant compared with the control group(P＜0.05),but the expression of CyclinD1 was reinforced(p＜0.01).Conclusions:The REGγgene promotes cell proliferation and inhibits apoptosis in breast cancer cells.The mechanism may be relevant to REGγparticipation in regulation of cell proliferation and apoptosis Cell cycle,ApoptosisPART THREEEFFECT OF TRANSFECTION OF REG./GENE ON THE GROWTH OF XENOGRAFTED HUMAN BREAST CANCER IN NUDE MICEObjective:To study the tumorigenesis of human breast cancer MDA-MB-231 cells in nude mice after transfection with REGγgene(a kind of proteasome activating factor) and the related mechanism.Methods:REGγgene was transfected into MDA-MB-231 cells in pcDNA3.1 vector via lipofectin mediation.The stable clones were selected by G418 600mg/L.The cells without transfection and transfected with empty vector were regarded as control groups.Three groups of cells were inoculated subcutaneously into nude mice.The growth of transplanted tumors was observed and the tumor inhibition ratio was calculated.The expression of REGγgene in tumor tissues was detected by RT-PCR.The expression of CD16 in tumor-infiltrating lymphocytes(TIL) and the cell cycle and apoptosis were determined by flow cytometry.The expression of p21 in transplanted tumors was examined by immunohistochemistry.Results.As compared with control group,xenografted tumor in REGγgene transfection group showed faster growth speed,larger tumor volume, and increased tumor weight(p＜0.05).The expression of REGγgene in tumors was markedly increased(p＜0.01).The expression of CD16 in TIL was greatly decreased(p＜0.05) and the cell proportion in G₀/G₁ phase and G₂/M phase decreased significantly but the cell percentage in S phase was obviously increased.The apoptotic ratio and the expression of p21 were obviously reduced(p＜0.05).Conclusions:REGγgene promotes the tumorigenesis and progress of breast cancer cells in nude mice.The mechanism may be explained by its effects of accelerating cell cycle,inhibiting apoptosis,suppressing the activation of NK cells,and specifically degrading p21.