Effects Of MSC-miRNA198-Exos On Proliferation,migration And Invasion Of Lung Adenocarcinoma Cells

Objective: miRNA is a genetic material that regulates gene expression,and is involved in cell proliferation,differentiation,aging,and apoptosis.Mesenchymal stem cells(MSCs)are adult stem cells with self-renewal and multidirectional differentiation potential that are involved in the process of tissue damage repair and regeneration.Studies have shown that MSCs exert a therapeutic effect mainly through the exosomes they secrete.Exosomes become the media of intercellular information transmission,participate in the proliferation,metastasis,invasion and angiogenesis of tumor cells,and play a key role in the occurrence and development of tumors.In this experiment,the miR-198 gene was transferred into human umbilical cord-derived mesenchymal stem cells by retroviral vectors,and their exosomes were extracted by ultracentrifugation,and the overexpressed miR-198 mesenchymal stem cell exosomes(MSC-miR-198-exo)were constructed to study the effects of human umbilical cord-derived MSC-miRNA198-exos on the proliferation,migration and invasion of lung adenocarcinoma cells.Methods: The overexpressed miR-198 retroviral vector(pMXS-hsa-miR-198-GFPuro#7)was transferred to human umbilical cord-derived mesenchymal stem cells(MSCs)by retroviral transfection method,and the transfection efficiency of miR-198 was observed by fluorescence microscopy,and the expression level of miR-198 in MSC was verified by qT-PCR.Mesenchymal stem cell exosomes(MSC-exo)and mesenchymal stem cell exosomes transfected with miR-198(MSC-miR-198-exo)were extracted by ultracentrifugation,Western blot detected exosome surface-specific markers CD9,CD63,TSG101,and analyzed exosome morphology and size by electron microscopy and particle size;qT-PCR was used to verify the expression levels of miR-198 in MSC-exo and MSC-miR-198-exo,and ms C-exo,A549 cell membranes and nuclei were labeled using fluorescent markers such as PKH26,Cell Mask,and Hoechest 33342,respectively,and fluorescence microscopy was used to observe exosome uptake by tumor cells.Tumor cells were intervened by MSC and MSC-miR-198-derived supernatants and exosomes,respectively,and the proliferation ability of tumor cells in vitro was detected by CCK-8 experiments;The Transwell experiment was used to analyze the invasion ability of tumor cells;the statistical analysis was based on SPSS22.0 statistical software and Graph Pad8.0 software for mapping,and P<0.05 believed that the difference was significant and statistically significant.Results: 1.Exosomes extracted by ultracentrifugation were analyzed by transmission electron microscopy and particle size,and the morphology was cup-like,and the size was about40-160 nm of bimeal vesicle-like structure.Western Blot results showed that the surface-specific markers CD9,CD63 and TSG101 were expressed in MSC-exo.qT-PCR results showed that the expression level of miR-198 in MSC cells(MSC-miR-198)transfected with miR-198 and exosomes(MSC-miR-198-exo)was significantly increased(P<0.01,P<0.01).3.Fluorescence microscopy observed that the red fluorescent marker-labeled exosomes were uptaken into the cell by A549 cells,indicating that A549 cells successfully uptake free MSC-exo from the extracellular environment.4.CCK-8 experimental results showed that MSC-miR-198-exo and MSC-miR-198-CM both inhibited the proliferation capacity of A549 cells(P<0.001,P<0.001);the results of scratches and Transwell cells showed that MSC-miR-198-exo and MSC-miR-198-CM inhibited the migration and invasion ability of A549 cells(P<0.01,P<0.001,P<0.001,P<0.001),the difference was statistically significant compared with the normal group,the MSC-exo group,or the MSC-CM group.Conclusion:The overexpression of miR-198 MSC-supernatants(MSC-miR-198-CM)and exosomes(MSC-miR-198-exo)intervened in A549 cells,which inhibited the proliferation,migration and invasion of tumor cells.This result shows that MSC-miR-198 delivers miR-198 through exosomes,thereby inhibiting the proliferation,migration and invasion of A549 cells.miR-198 may be a class of tumor suppressor genes,and MSC-exo may also become a new type of biological carrier,providing new ideas for lung cancer patients to find new gene targets and treatments.