The Study Of Intercellular MRNA Transfer Medicated By Cell-in-cell Structure

BackgroundThe transfer of mRNA between mammalian cells has been found for a long time.Due to the lack of effective research models and means,the mechanism of mRNA transfer between cells and whether mRNA is translated into proteins and plays its biological functions after reaching the receptor cells are still unknown.Cell-in-cell is a special cell structure that one or more live cells drill into another live cell.The main fate of drilling cells(effector cells)is death(intracellular death)in the drilled cells(target cells).Most research has focused on the mechanism of cell-in-cell and the death type of internal effector cells,for the study of the external target cells fate and whether the biological molecules,such as mRNA transfered between effect cells and target cells is limited for a long time due to the lack of research model.ObjectiveBased on the Cre-loxP enzyme system,this project intends to take cell-in-cell structure as the research model to study the transmission of intercellular mRNA and its biological function in target cells,as well as investigate the role of cell-in-cell in cell communication.MethodsPlvx-IRES-Neo-Cre lentivirus recombinant plasmid was constructed to package the virus in HEK293 FT cells and infect different epithelial cell lines as well as tumor cell lines.After antibiotic screening,cells with high Cre expression were screened and identified by immunofluorescence or western blot.Then,monoclonal cell lines(effector cells)expressing Cre were established.The monoclonal cell lines(target cell)carrying theloxP-DsRed-loxP-eGFP sequence was constructed.and the target cells were transfected with pcDNA3.1-CMV-CFP;UBC-Cre25 nt plasmid to see whether they could be recombined(express the fluorescence signal of eGFP).Effector cells were co-cultured with target cells,the target effector cells co-cultured without contact by transwell and the target cells cultured by effector cell supernatant were set as controls.After 72 hours,the expression of eGFP signal in each group was observed and counted.The effects of effector cell death,cell membrane nanotubes,and Rho-ROCK signaling pathway on the transmission of Cre mRNA were analyzed.Using flow cytometry sorting out the cell-in-cell structure after co-culture,the cells without cell-in-cell structure sorted by flow cytometry and co-culture cells without flow cytometry set as controls.After 72 hours,the expression of eGFP signal in each group was observed and counted,analysis of the role of cell-in-cell structure in mediating target cells recombination.The process of expressing eGFP signal in target cells that occur cell-in-cell structure was recorded by time-lapse.ResultsMCF7 breast cancer cell lines and PLC/PRF/5 cell lines with stable expression of loxP-DsRed-loxP-eGFP sequence,as well as MCF7 breast cancer cell lines and MCF10 A cell lines with stable expression of Cre were successfully constructed.After co-culture 72 hours of the effective-target cells,it was observed that the eGFP signal is positive in co-culture cells while the transwell group and target cells with effector cells supernatant were negative,indicating that intercellular mRNA transfer depended on cell-cell contact.The expression rate of eGFP signal in the group with cell-in-cell structure after flow cytometry sorting was significantly higher than that of the effector target cells co-culture group without flow cytometry sorting,suggesting that cell-in-cell is an important pathway to mediate gene recombination in the target cells.The results of inducing effector cell death showed that the transmission of Cre mRNA was mainly through living cells.Membrane nanotubes and Rho-ROCK signal pathway inhibitors can partially block the transfer of Cre mRNA.The process of target cell genome recombinationand expression of eGFP signal was successfully captured by time-lapse,which proved that cell-in-cell can regulate the transfer of mRNA between cells,and the mRNA from effector cells can be translated into proteins in the target cells to change the biological characteristics of the target cells.ConclusionmRNA can be transferred in living cells through cell-cell contact,and can be translated into proteins after entering target cells to play biological functions,cell-in-cell is a new way to mediate intercellular information communication.