Abstract:

Objective To discuss the effect of exosome （Exo） microRNA-761 （miR-761） on the epithelial-mesenchymal transition （EMT） process of the osteosarcoma （OS） cells by regulating tumor-associated macrophage （TAM） polarization， and to clarify its related mechanism. Methods The miR-761 plasmid and negative control （miR-NC） plasmid were transfected into the HEK293 cells， and the non-transfected cells were regarded as control group. The transfection efficiency was detected using real-time fluorescence quantitative PCR （RT-qPCR） method.The Exo containing miR-761 was isolated， and the morphology of Exo was observed by transmission electron microscope. The concentration and size distribution of Exo samples were detected by nanoparticle analyzer， and the expression of Exo surface marker protein was detected by Western blotting method.The human monocyte leukemia THP-1 cells were stimulated with phorbol 12-myristate 13-acetate （PMA） to become the M0 macrophages， which were then treated with Exo containing miR-761 and co-cultured with the OS MG63 cells to establish the co-culture system. The experiment was divided into M0 group， TAM group， miR-761 NC group， and miR-761 Exo group. The M0 macrophages were collected from various groups， and the positive rates of M1 macrophage marker CD86 and M2 macrophage marker CD206 in various groups were detected by flow cytometry； the protein expression levels of M1 macrophage secreted factors interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） and M2 macrophage secreted factors interleukin-10 （IL-10） and transforming growth factor-β1 （TGF-β1） in various groups were detected by Western blotting method. The M0 macrophages were treated with Exo containing miR-761 and co-cultured with MG63 cells to establish the co-culture system. The experiment was divided into control group， TAM group， miR-NC Exo+TAM group， and miR-761 Exo+TAM group. The MG63 cells in various groups were collected， and the fluorescence intensities of E-cadherin and Vimentin in the MG63 cells in various groups were observed by immunofluorescence staining； the expression levels of E-cadherin， Vimentin， and EMT regulation-related transcription factors Twist1， Snail， and Slug proteins in the cells in various groups were detected by Western blotting method；the numbers of invasion and migration cells in various groups were detected by Transwell chamber assay. Results The HEK293 cells containing miR-761 were successfully obtained by transfection experiments， and the Exo was isolated. Compared with M0 group， the positive rate of CD86 of the macrophages in TAM group was decreased （P<0.05）， while the positive rate of CD206 was increased （P<0.05）， the expression levels of IL-1β and TNF-α proteins were decreased （P<0.05）， while the expression levels of IL-10 and TGF-β1 proteins were increased （P<0.05）. Compared with TAM group， the positive rate of CD86 of the macrophages in miR-761 Exo group was increased （P<0.05）， while the positive rate of CD206 was decreased （P<0.05）， the expression levels of IL-1β and TNF-α proteins were increased （P<0.05）， while the expression levels of IL-10 and TGF-β1 proteins were decreased （P<0.05）. Compared with control group， the fluorescence intensity of E-cadherin in the MG63 cells in TAM group was decreased， while the fluorescence intensity of Vimentin was increased， the expression level of E-cadherin protein was decreased （P<0.05）， while the expression levels of Vimentin， Twist1， Snail， and Slug proteins were increased （P<0.05）， and the numbers of invasion and migration cells were increased （P<0.05）. Compared with TAM group， the fluorescence intensity of E-cadherin in the MG63 cells in miR-761 Exo+TAM group was increased， while the fluorescence intensity of Vimentin was decreased， the expression level of E-cadherin protein was increased （P<0.05）， while the expression levels of Vimentin， Twist1， Snail， and Slug proteins were decreased （P<0.05）， and the numbers of invasion and migration cells were decreased （P<0.05）. Conclusion The exo-delivered miR-761 can inhibit the EMT process of the OS cells， thereby inhibiting the cell migration and cell invasion； its mechanism may be related to regulating TAM polarization.

Key words: Osteosarcoma, MicroRNA-761, Exosome, Epithelial-mesenchymal transition, Tumor-associated macrophage polarization