Exosomal miR-767 from senescent endothelial-derived accelerating skin fibroblasts aging via inhibiting TAB1

Abstract

Skin aging is a complicated physiological process, and microRNA-mediated regulation has been shown to contribute to this process. Exosomes mediate intercellular communication through miRNAs, mRNAs and proteins, and participate in many physiological and pathological processes. Vascular endothelial cell-derived exosomes have been confirmed to be involved in the development of many diseases, however, their effects on skin aging have not been reported. In this study, senescent endothelial cells could regulate skin fibroblast functions and promote cell senescence through exosomal pathway. miR-767 was highly expressed in senescent vascular endothelial cells and their exosomes, and miR-767 is also upregulated in skin fibroblasts after treatment with exosomes derived from senescent vascular endothelial cells. In addition, transfection with miR-767 mimic promoted senescence of skin fibroblasts, while transfection with miR-767 inhibitor reversed the effect of D-galactose. Double luciferase analysis confirmed that TAB1 was a direct target gene of miR-767. Furthermore, miR-767 expression was increased and TAB1 expression was decreased in D-galactose induced aging mice. In mice that overexpressed miR-767, HE staining showed thinning of dermis and senescence appearance. In conclusion, senescent vascular endothelial cell-derived exosome mediated miR-767 regulates skin fibroblasts through the exosome pathway. Our study reveals the role of vascular endothelial cell-derived exosomes in aging in the skin microenvironment and contributes to the discovery of new targets for delaying senescence.

Keywords: Aging; Exosome; Fibroblasts; TAB1; miR-767.

Fig. 1

Staining of senescent cells. A SA-β-gal staining and positive cell number analysis of endothelial cells induced by D-gal for 48 h (200 ×). B SA-β-gal staining and positive cell number analysis of fibroblasts cells induced by D-gal for 48 h (100 ×). C SA-β-gal staining and positive cell number analysis of skin fibroblasts after co-culture with aging endothelial cells (Model) and GW4869 after co-culture (Model + GW) (100 ×)

Fig. 2

miR-767 expression in exosomes of senescent endothelial cells and regulates senescence in fibroblasts. A Expression of miR-767 in skin fibroblasts by qRT-PCR. B Expressions of miR-767 in vascular endothelial cells by qRT-PCR. C Expression of miR-767 in skin fibroblasts after co-cultured with senescent endothelial cells by qRT-PCR. D Exosomes derived from vascular endothelial cells were observed by ultraelectron microscopy. E Expression of miR-767 in exosomes derived from senescent vascular endothelial cells by qRT-PCR. F SA-β-gal staining and Hoechst staining of skin fibroblasts with senescent vascular endothelial cells derived exosomes (100 ×). G Expression of miR-767 after transfection with miR-767 mimic and miR-767 inhibitor in skin fibroblasts by qRT-PCR. H The ratio of positive cells in skin fibroblasts after transfection miR-767 mimic was detected by SA-β-gal staining (100 ×). I The ratio of positive cells in skin fibroblasts after transfection miR-767 inhibitor + D-gal was detected by SA-β-gal staining and Hoechst staining (100 ×)

Fig. 3

TAB1 affects senescence related biological functions of skin fibroblasts. A Expression of TAB1 after transfection with miR-767 mimic and miR-767 inhibitor in skin fibroblasts by qRT-PCR. B Schematic diagram of binding of mmu-miR-767 to m-TAB1-3UTR target sites. C The interaction between mmu-miR-767 and m-Tab1-3UTR was detected by Double Luciferase reporter gene. D Ratio of positive cells in siTAB1-treated skin fibroblasts stained with SA-β–gal staining (100 ×), Hoechst staining (100 ×) and Ki67 staining (200 ×)

Fig. 4

Analysis of miR-767 and TAB1 in aging skin. A HE staining for D-gal induced skin tissue(100 ×). B Expression of miR-767 in D-gal induced skin tissue by qRT-PCR. C Expression of TAB1 in D-gal induced skin tissue by qRT-PCR. D Expression of TAB1 in D-gal induced skin tissue by WB. E HE staining and Masson staining for miR-767 tagomir induced skin tissue(100 ×). F Expression of miR-767 in miR-767 tagomir induced skin tissue by qRT-PCR