Construction Of MiR-21-4T1Cells And Functional Studies Of Its Exosomes

Objective: microRNA-21（miR-21）plays an important role in tumordevelopment and progression, which has a high-expression in many solidtumors, including the breast tumor. Exosomes are a subcategory of bilayermembrane-bound nanovesicles released from normal and diseased cells, theycontain and carry many proteins, mRNAs and microRNA species.Cancershave adapted the exosome as a pathway by which neoplastic cellscommunicate with each other (autocrine) and with nonneoplastic cells(paracrine and endocrine); via this pathway, cancer suppresses the immunesystem and establishes a fertile local and distant environment to supportneoplastic growth, invasion, and metastases. For the futher reaserch on thequestion whether microRNA-21can contribute significantly to cancer growthand development, we construct the miR-21-4T1cell strain, and the explorationhad been made for the study of its founction on4T1cells and immune cells.Methods:1The construction of miR-21-4T1cell strainWe constructed miR-21-4T1cell strain and scramble-4T1cell strain withthe GeneCopoeia HIV-Based Lentiviral Expression System. The expressionlevel of miR-21in miR-21-4T1cell was detected by real-time PCR. Thefluorescent microscopy and Flow cytometry was used to analysis theexpression of green fluorescent proteins in miR-21-4T1cell andscramble-4T1cell.2The effects of miR-21on the biological characters of4T1cell in vitroThe influnce of miR-21on4T1cell proliferation was accessed by MTSassay. The effect of miR-21on the migration capacity of4T1cell was detectedby Wound healing and Transwell chamber assay.3Preparation of exosomes For preparation of exosomes, cells were cultured in vitro at37°C in ahumidified5%CO2atmosphere, and the supernatants were harvested after72hin culture. The exosomes were purified from the supernatants by differentialcentrifugation. In brief, cells were removed by centrifugation for10min at300×g;20min at2,000×g;30min at10,000×g. Supernatants were collectedand centrifuged for120min at100,000×g. Exosomes were pelleted at thefinal centrifugation step and were resuspended in PBS, then they were stainedwith phosphotungstic acid solution.Transmission electron microscopy wasused to observe their ultrastructures.4Founction studies of exosome derived from miR-21-4T1cellThe expression level of miR-21in exosome derived from miR-21-4T1cell was detected by real-time PCR. The influnce of exosome secreted bymiR-21-4T1cell on4T1cell proliferation was accessed by MTS assay, andthe effect of exosome secreted by miR-21-4T1cell on the migration ability of4T1cell was detected by Wound healing and Transwell chamber assay. Thelevel of IL-6，IL-10pruduced by murine bone marrow-derived dendritic cellsor mouse peritoneal macrophage,which were cultured with the exosomederived from miR-21-4T1cell,were analyzed by ELISA test. Effect ofmiR-21-4T1exosomes on the expression of DC surface makers was detectedby Flow cytometry.Results:1Real-time PCR results showed that miR-21was up-regulated2.79±0.5411folds in miR-21-4T1cell. Under the fluorescent microscopy, the greenfluorescence was spread in the scramble-4T1cells and miR-21cells, meaningthe eGFP was expressed. Flow cytometry analysis revealed that theproportions of eGFP+cells of scramble-4T1and miR-21-4T1were77.06%±0.844and93.58%±0.456.2MTS assay showed that there was no significantly statistical differencewhen miR-21was up-regulated. The migration ability of miR-21-4T1cell washigher than4T1cell deduced from the Wound healing and Transwell chamberassay. 3Transmission electron microscopy revealed that exosomes from tumorcell culture supernatants were40to100nm membrane vesicles. They wereround or ellipse in shape.4Real-time PCR analysis revealed that miR-21in exosome derived frommiR-21-4T1cell was up-regulated3.49±0.2703folds.5MTS assay and the Wound healing assay display that there were nosignificantly statistical differences between the three groups. Howeverexosome secreted by miR-21-4T1cell increased the migration ability of4T1cell in the Transwell chamber assay.6Exosome derived from miR-21-4T1cell increased the production ofIL-6，IL-10by mouse peritoneal macrophage,and IRS661had no influence onthis result.7Exosome derived from miR-21-4T1cell increased the production ofIL-6，IL-10by murine bone marrow-derived dendritic cells, and decreased theexpression of MHC II on murine bone marrow-derived dendritic cells.Conclusion:1MiR-21increases the migration ability of4T1cell.2The expression of miR-21was up-regulated in the exosome derivedfrom miR-21-4T1cell.3Exosome derived from miR-21-4T1cell increase the migration abilityof4T1cell, but it didn’t increase the proliferation of4T1cell.4TD-exosome suppress the immune cells, however exosome derivedfrom miR-21-4T1cell didn’t increase this effect.