Effects Of Exosomes Riched In MiR-302a Derived From HUCMSCs On EC

ObjectiveThe purpose of this study was to verify a potential tumor suppressor microRNA,miR-302 a,and engineer hUCMSCs to secrete exosomes enriched with miR-302 a for therapy of endometrial cancer.Methods1.By analyzing theexpression of miRNAs from EC tissue and their matching adjacent tissue samples to acquire miR-302 a as asociated with the developmen of EC,and to study the antitumor activities.2.Isolation and identification of hUCMSCs: Umbilical cord samples were collected from full-term births after normal or cesarean delivery,and hUCMSCs were isolated by tissue block attachment method.Then cells cultured with α-MEM medium containing 10% fetal bovine serum.Detection of hUCMSCs surface markers,like CD73,CD90 and CD105,were assessed by FACScaliber Flow Cytometer.And meanwhile we conducted the adipogenic and osteogenic differentiation experiments.3.Isolation of exosomes riched in miR-302a: We established miR-302a-overexpressing cells in hUCMSCs using lentivirus transfection.Then the exosomes riched in miR-302 a with common exosomes markers(CD63、CD81 and HSP70)were isolated from the culture supernatant using the ExoQuick-TC kit.The morphology of the exosomes was observed using transmission electron microscopy.ZetaView was used to detect nanoparticles of the size and distribution or concentration of exosomes.4.The effects of exosomes rich in miR-302 a derived from hUCMSCs on the proliferation and migration of EC cells(Ishikawa and ECC-1),and the expression of Cyclin D1 and AKT.Results1.Our data showed that miR-302 a expression was downregulated in EC tissues compared with adjacent tissues and proliferation and migration capacity of ISK and ECC-1 cells were significantly suppressed when overexpressed miR-302 a in these cells.2.We successfully isolated hUCMSCs from human umbilical cord Wharton’s jelly using tissue adherence method.These hUCMSCs express mesenchymal antigens,which are positive for CD73,CD90 and CD105,while lack hematopoietic marker CD45.We used the third generation of hUCMSCs to perform osteogenic differentiation,stained with Alizarin Red S for positive detection.Adipocytic phenotypes of induced hUCMSCs were signaled by the appearance of tiny intracytoplasmic lipid droplets in cells,and lipid granules tended to unite and were stained with oil red O.3.We used ExoQuick-TC kit to isolate exosomes from the culture medium of hUCMSCs,then visualized by transmission electron microscopy.The results showed typical rounded particles ranging from 40-200 nm in diameter.Nanoparticle tracking analysis(NTA)characterized the size of exosomes derived from hUCMSCs at a peak of 108 nm.Western blotting analysis demonstrated the presence of exosomes markers such as CD63、CD81 and HSP70.4.Our results provided strong evidence that miR-302a-enriched exosomes caused a significant reduction in proliferation and migration of EC cells by downregulating Cyclin D1 through an interaction with AKT,a cell cycle regulator.Conclusions1.Overexpression of miR-302 a inhibits EC cells proliferation and migration.2.Exosomes derived from hUCMSCs can act as an effective carrier of miRNA,which can transfer specific miRNA(miR-302a)to EC cells.3.Exosomes rich in miR-302 a inhibit Cyclin D1 and AKT expression in EC cells.