外泌体荷载miR-122靶向ADAM10调控肝癌HepG-2细胞生物学活性的机制研究OA

Objective:To investigate the regulatory effect and molecular mechanism of microRNA(miR-122)delivered by bone marrow mesenchymal stem cell(BMSC)-derived exosomes(Exo)on the biological activity of human HepG-2 liver cancer cells.Methods:Exo were isolated from BMSCs by ultraspeed gradient centrifugation and identified by transmission electron mi-croscopy and Western blot.The miR-122 mimics and its negative control(NC mimics)were loaded into Exo by electrofection,re-spectively,and recorded as NC Exo and miR-122 Exo,respectively,the relative expression of miR-122 in the two types of Exo was detected by RT-qPCR.NC Exo and miR-122 Exo were labeled with exosome green fluorescent PKH67 dye and then co-cultured with HepG-2 cells to observe the uptake of Exo by HepG-2 cells.HepG-2 cells were divided into the control group,the NC Exo group(NC Exo-treated cells),and the miR-122 Exo group(miR-122 Exo-treated cells).CCK-8 method,Transwell chamber experiment,Western blot experiments were used to detect the proliferation activity,migration and invasion ability,the expression changes of E-cadherin,N-cadherin and Vimentin of HepG-2 cells in each group.Bioinformatics and dual luciferase re-porter gene experiments predicted and verified the targeted regulatory relationship between miR-122 and a disintegrin metallopro-teinase 10(ADAM10).Results:Exo isolated from BMSCs were spherical vesicles with a diameter ranging from 30 nm to 150 nm,Exo special marker proteins CD9,CD63,HSP70 and TSG101 were all positively expressed,the relative expression of miR-122 in miR-122 Exo loaded with miR-122 mimics was significantly higher than that in NC Exo loaded with mimics NC(P<0.05),both NC Exo and miR-122 Exo could be taken up by HepG-2 cells.Compared to the control group,the proliferation activi-ty of HepG-2 cells in the NC Exo group at 24,48,and 72 h of culture were significantly increased(P<0.05),and the number of cell migration and invasion was significantly increased(P<0.05).The expression level of epithelial marker E-cadherin protein in HepG-2 cells was significantly reduced(P<0.05),while the expression levels of stromal markers N-cadherin and Vimentin pro-tein were significantly increased(P<0.05).Compared to the control group and the NC Exo group,the proliferation activity of HepG-2 cells in the miR-122 Exo group significantly inhibited at 24,48,and 72 hours of culture(P<0.05),and the number of cell migration and invasion was significantly reduced(P<0.05).The expression of E-cadherin protein in miR-122 Exo HepG-2 cells was significantly increased(P<0.05),while the expression levels of N-cadherin and Vimentin proteins were significantly de-creased(P<0.05).There is a binding site between miR-122 and the 3'-UTR of ADAM10.Compared to the miR-NC group,transfection of miR-122 in HepG2 cells can significantly inhibit the relative luciferase activity of ADAM10 WT(P<0.05).Con-clusion:Delivery of miR-122 to HepG-2 cells by Exo can inhibit cell proliferation,migration,invasion,and epithelial-mesenchymal transition,and its mechanism may be related to the targeted inhibition of ADAM10 expression.