Research On The Invasive Phenotypes Of Ovarian Cancer Mediated By Exosomes

BackgroundOvarian cancer(OC)is the most lethal gynecological neoplasm with an overall 5-year survival rate of 47%during 2018.These poor outcomes are due in part to the fact that OC exhibits early invasion and rapid metastasis.Exploring the detailed metastatic mechanisms of OC may facilitate the identifications of novel diagnostic molecular markers or therapeutic targets.Tumor heterogeneity has been widely described and considered one of the main factors that can accelerate the tumor evolution influencing the metastatic potential and therapeutic resistance.Within the complex context of a heterogeneous tumor,sub-clones with distinct characteristics may establish positive interactions resulting in a phenotypic switch that promotes tumor growth and metastasis.Some interesting researches mentioned that tumor-derived exosomes(TDE),which play an important role in cellular communication,may take part in these interactions.As known to all that heterogeneity is one of characteristics in OC,in this study,we put forward a hypothesis about whether less aggressive ovarian cancer cells exhibit increased migratory abilities after uptaking exosomes generating by highly aggressive ovarian cancer cells and further consider the molecular mechanisms underlying this phenomenon.ObjectiveTo investigate the detailed roles and mechanisms of tumor-derived exosomes in progression and metastasis of OC in vitro.Materials and methodsFirst,transwell chambers assay and cell proliferation recording were separately conducted to show the characterizations of HO8910 and HO8910PM cells;HO8910PM and HO8910-derived exosomes were isolated by differential centrifugation method;the morphology,size and biological markers of exosomes were separately defined by transmission electron microscopy,nanoS90 and Western blotting;HO8910 cells were able to uptake the exosomes labeled with fluorescent dye PKH67,which can be tracked under confocal microscopy;Transwell chambers assay and cell proliferation recording were used to compare whether HO8910PM-derived exosomes influence the migration and invasion of HO8910 cells;to explore the molecular mechanisms further,RNA seq was used to find the different expressed genes between these two cell lines,then the expression level of FN1 between HO8910PM and HO8910 was validated by real time PCR.Accordingly,Fibronectin encoded by FN1 was measured by ELISA.Lastly,real time PCR and ELISA was used to verify whether exosomes originating from HO8910PM cells influence expression level of FN1 in HO8910 cells.Meanwhile,Enforced modulating the expression of FN1 significantly influenced the migration and invasion of HO8910PM cells.Results1.Differential centrifugation obtained exosomes from cell supernatant successfully.2.HO8910PM-generated exosomes promoted migration and invasion of H08910 cells.3.RNA sequencing was carried out in HO8910PM and HO8910 cells.476 differentially expressed genes was discovered,including 411 up-regulated genes and 65 down-regulated genes in HO8910PM cells.Real time PCR confirmed that FN1 was up regulated in HO8910PM cells.ELISA proved that Fibronectin encoded by FN1 was up regulated significantly in the cell supernatant of HO8910PM compared to HO8910.4.FN1 knockdown resulted the decrease of Fibronectin in the cell supernatant and inhibited migration and invasion in H08910PM cells.5.Real time PCR validated that HO8910PM-generated exosomes increased the expression level of FN1 in HO8910 cells.Accordingly,ELISA confirmed that HO8910PM-generated exosomes increased the expression level of Fibronectin in the cell supernatant of HO8910 cellsConclusion1.Differential centrifugation obtained exosomes from cell supernatant successfully.2.Exosomes derived from cells with high metastatic potential can modulate phenotypic plasticity in less aggressive cancer cells and elicit these alterations through up regulating FN1 expression.