The Function Research Of ChPrP~C Involved In The Processes Of DF-1Cells Proliferation And Adhesion

【Objective】Using green fluorescent protein as a reporter gene, the eukaryotic expression vectors wereconstructed by cloning the poultry source U6(cU6) and human source U6(hU6) promoter in this study. cU6and hU6promoter transcription activity were compared respectively in HeLa and DF-1cells.The potential ofcU6promoter used in vitro expression system of avian cells was analyzed.The eukaryotic expression vectorscontaining cU6promoter were constructed and the cell lines with over-expression chickens prion (ChPrPC) ofDF-1were established,which provides a stable cell model for further studying the effect of ChPrPCin aviancell differentiation, proliferation and apoptosis.The effect of over-expression ChPrPCin DF-1cellsproliferation, adhesion and invasion was analyzed reference to the function of PrPCin promoting cellproliferation and adhesion, which would provide the basis for further elucidating the physiological functionsof ChPrPC.【Methods】The specific primers were designed according to the reported U6promoter sequence. Then cU6and hU6core promoter sequences were obtained by PCR amplification and cloning. Using green fluorescentprotein as a reporter gene, the recombinant eukaryotic expression vectors were constructed by cloned cU6and hU6core promoter sequences. The Hela and DF-1cell lines were transfected with cU6and hU6recombinant expression vectors. The fluorescence positive cells and EGFP expression levels of HeLa andDF-1cells were detected after transfected12,24and36h by inverted fluorescence microscope, real-timequantitative RT-PCR, Western blot and flow cytometry. The primers were designed according to the chickenprion gene full-length sequence cloned in our laboratory. Chicken prion gene ORF were obtained by PCRamplification and cloning, ChPrPCrecombinant expression vectors were constructed with high transcriptionalactivity cU6promoter. The DF-1cell lines with over-expression ChPrPCwere established by G418selection.ChPrPCexpression levels of the selected DF-1cell lines were detected by Western blot and real-timequantitative RT-PCR.The effect of over-expression ChPrPCin DF-1cells proliferation, adhesion and invasionwas analyzed by cell adhesion assay, invasion assay, MTT and flow cytometry.【Results】(1) About420bp,464bp and392bp expected fragment of hU6, cU6-3and cU6-1promotersequences were obtained by PCR amplification from a template containing Hela or DF-1cell genomic DNAused three pairs of specific primers. The target fragments undergone double enzyme digestion and insertedrespectively in the pEGFP-N1vector.Then the recombinant expression vectors of pEGFP-N1-cU6-1,pEGFP-N1-cU6-3and pEGFP-N1-hU6were successfully constructed,which respectively transfected intoHela and DF-1cells.The promoter transcriptional activity test results show that EGFP expression levels andfluorescence-positive cell number mediated by three promoters were gradually increased from transfected12h to36h in both cell lines, but EGFP mRNA transcription levels were gradually decreased.The transcriptional activity of pEGFP-N1-cU6-1and pEGFP-N1-cU6-3was significantly higher thanpEGFP-N1-hU6（P<0.01）in DF-1cell. The transcriptional activity of pEGFP-N1-hU6was highest in Helacell.And in both cell lines, the mRNA transcription levels and fluorescence intensity of EGFP mediated bypEGFP-N1-cU6-1were higher than pEGFP-N1-cU6-3.(2)Chicken prion gene were cloned successfully,about858bp and860bp. The sequences were inserted in pcDNA3.0and pEGFP-N1vector containingcU6-1promoter and the recombinant expression vectors of pCDNA-cU6-1-ChPrP andpEGFP-N1-cU6-1-ChPrP were successfully constructed. The DF-1cell lines with over-expression ChPrPCwere established by pCDNA-cU6-1-ChPrP and pEGFP-N1-cU6-1-ChPrP vectors transfected. ChPrPCexpression levels of the selected DF-1cell lines were detected by Western blot and real-time quantitativeRT-PCR.The results show that ChPrPCexpression levels of DF-1-PrP and DF-1-EGFP-PrP were significantlyincreased, about60and70times compared control cells.(3) The cell adhesion and invasion assay resultsshowed that the adhesion and invasion ability of DF-1-PrP on extracellular matrix was significantly higherthan the empty vector cells DF-1-cont(P <0.01),and there was no significant difference between DF-1-contand normal DF-1cell. The flow cytometry and MTT assay results show that the apoptosis rates of DF-1-PrP，DF-1-cont and DF-1cell were1.7%±0.06%,10.24%±0.11%and11.75%±0.24%, DF-1-PrP apoptosiswas significantly lower than the other two groups of cells (P <0.01), The proliferation rate of DF-1-PrP cellswas also significantly faster than the DF-1and DF-1-cont cells.【Conclusions】(1)The recombinant avian origin U6promoter expression vectors of pEGFP-N1-cU6-1,pEGFP-N1-cU6-3and pEGFP-N1-hU6were successfully constructed,and the transcriptional activity ofcU6-1promoter was highest. cU6-1promoter can be used for vitro expression vector construction in poultrycells, which would provide a platform for foreign gene expression in avian cells in the future.(2) The DF-1cell lines of pEGFP-N1-cU6-1-ChPrP and pCDNA-cU6-1-ChPrP with over-expression ChPrPCwereestablished,which provides conditions for further researching the molecular mechanisms of ChPrPCeffect inDF-1cells proliferation, adhesion and invasion.(3)ChPrPCinvolved in the processes of DF-1cellsproliferation, adhesion and invasion, which could promote DF-1cell proliferation, slow down DF-1cellapoptosis and improve DF-1cell adhesion and invasion ability to the extracellular matrix. This resultsprovides references for further researching the molecular mechanisms of ChPrPCpromoting DF-1cellsproliferation, adhesion and invasion,which may help to elucidate the physiological function of ChPrPC.