The Mechanism Of Exosomal MiR-499a-5p Regulating The Metastasis In Lung Adenocarcinoma

ObjectiveThe exosomes are 30-150 nm sized extracellular microvesicles,containing biologically active components such as functional mRNAs and miRNAs.Exosomes can be absorbed and secreted by different types of cells,especially tumor cells.Tumor-derived exosomes can serve as delivery vehicles for miRNAs and promote the proliferation,migration,invasion,and epithelial-mesenchymal transition when co-cultured with tumor cells,eventually contributing to tumor metastasis.However,the specific mechanism remains unclear.The aim of this study was to investigate the biological functions of exosomal miRNAs in the development and metastasis of lung adenocarcinoma,and to elucidate the underlying molecular mechanisms,thus to provide new therapeutic options for metastatic lung cancer.MethodsFirstly,exosomes were isolated from the culture medium of three lung adenocarcinoma cells by ultracentrifugation,and the phenotype of exosomes was identified by transmission electron microscopy,nanoparticle tracking analysis,fluorescent staining,and Western blot.The expression levels of miR-499a-5p in different cells and their exosomes were detected by high-throughput sequencing and qPCR.Secondly,after the overexpression and knockdown of miR-499a-5p were conducted,CCK8,scratch,transwell migration assays,Western blot were performed to study the effect of miR-499a-5p on cell proliferation,migration and EMT in vitro.Subcutaneous lung cancer xenograft models were established to observe the effect of miR-499a-5p on tumor growth in vivo after injecting activators and inhibitors.To further explore the relevant molecular mechanisms,mTOR pathway inhibitor rapamycin and activator MHY1485 were introduced to verify whether miR-499a-5p influenced lung cancer through the mTOR pathway.Finally,non-/highly-metastatic tumor-derived exosomes were co-cultured with recipient lung cancer cells to observe the impacts on cell proliferation,migration and EMT.Meanwhile,cells were transfected with miR-499a-5p inhibitors to study whether the impacts could be inhibited.ResultsThis study revealed that the expression of miR-499a-5p was significantly up-regulated in exosomes derived from highly metastatic lung adenocarcinoma cells.Overexpression of miR-499a-5p promoted the proliferation,metastasis and EMT of lung cancer cells in vivo and in vitro,and knockdown of miR-499a-5p inhibited the above effects.Overexpression of miR-499a-5p activated the mTOR pathway,and rapamycin inhibited its effects on lung cancer cell migration.On the contrast,the knockdown of miR-499a-5p suppressed the mTOR pathway.Highly metastatic lung cancer-derived exosomes could serve as a vehicle for miR-499a-5p,facilitating the cell-to-cell communication.Mir-499a-5p sufficient exosomes promoted the proliferation,migration and EMT,and the process could be blocked by miRNA inhibitors.Discussion and ConclusionThis study revealed the correlation between exosomal miR-499a-5p and metastasis in lung adenocarcinoma,and established the existence of a miR-499a-5p/mTOR axis which could promote lung cancer development,metastasis and EMT in vivo and in vitro.It reveals the potential diagnostic and therapeutic value of tumor-derived exosomal miR-499a-5p and sheds a new insight on a novel molecular mechanism which modulates metastasis.