Exosome-derived TGF-β1-miR-411-5p-MAP3K1/SP2 Pathways Regulate Melanoma And Its Microenvironment

Objective : To explore the regulation mechanism of TGF-β1 — miR-411-5p —MAP3K1/SP2 pathways in melanoma(MM)and its microenvironment,and provide new therapeutic targets.Methods:(1)The expressions of TGF-β1were identified by Real-time PCR and western blot assays in melanoma and melanocytic nevi tissue in vivio.(2)The expressions of TGF-β1were identified by Real-time PCR and western blot assays in melanoma and melanocytic nevi cells in vitro.(3)The expressions of TGF-β1 were identified respectively by western blot assays in exosomes secreted by melanoma cell line A375 and melanocyte cell line HEM-L.(4)Melanoma-derived exosomes were co-cultured with normal human fibroblast cell line WS1.(5)The most widely and obviously different microRNA was selected by Real-time PCR.(6)MTT assay were conducted to determine cell proliferation in A375 and cancer associated fibroblasts(CAF)after transfection with miR-411-5p-mimics.(7)Correlation analysis among clinical pathologicparameters,TGF-β1 and miR-411-5p was carried on after immunohistochemical stain.(8)MiR-411-5p target genes were predicted by bioinformatics technology and network resources and validated by Real-time PCR and western blot assays.(9)Flow cytometry was used to detect the cell cycle of A375 transfected with MAP3K1 siRNA.(10)Phenotypic changes of CAF were observed after immunofluorescence staining.Results:(1)In vivo,the expression of TGF-β1 was increased in the parenchyma tissue of melanoma compared with that of melanocytic nevi.The expression of TGF-β1 was increased in the interstitial tissue of melanoma compared with that of melanocytic nevi.(2)In vitro,the expression of TGF-β1 was increased in A375 and the expression of TGF-β1 increased in CAF.(3)Compared with exosomes secreted by HEM-L,the expression of TGF-β1 was increased in exosomes secreted by A375.(4)Normal human fibroblast cell WS1 was transformed into cancer associated fibroblast after co-culture with exosomes secreted by melanoma cell line A375.(5)MiR-411-5p negatively correlated with TGF-β1 was the most widely and obviously different microRNA in MM cells and CAF.(6)Re-expression of microRNA-411-5p reduced the proliferation and invasion abilities of melanoma A375 cells,and reduced the invasion ability of CAF.(7)The expressions of TGF-β1 and microRNA-411-5p were correlated with pathological grade,depth of invasion and lymph node metastasis.(8)The target gene SP2 and MAP3K1 were significantly decreased in A375 cells after transfection with miR-411-5p-mimics.The target gene SP2 was significantly decreased in CAF after transfection with miR-411-5p-mimics.(9)Transfection of MAP3K1 siRNA into melanoma cell line A375 caused cell cycle arrest in G1 phase.(10)The expression of vimentin protein was decreased in the CAF transfected with SP2 siRNA.Conclusions1.Melanoma cells can secrete exosomes with a high level of TGF-β1.2.MiR-411-5p is negatively regulated by TGF-β1.3.Exosome-derivedn TGF-β1 can activate the cell cycle leading to melanoma cell proliferation through autocrine action and TGF-β1-miR-411-5p-MAP3K1 pathway.4.Exosome-derived TGF-β1 can participate adjacent fibroblasts transforming into the cancer associated fibroblasts and form tumor microenvironment through paracrine action and TGF-β1-mi R-411-5p-SP2 pathway.