The Effect Of USF1 On The Development And Progression Of Melanoma Through Regulating TGF? Signaling Pathway

Background:Upstream stimulatory factor 1 is a multifunctional transcription factor that is ubiquitously expressed in eukaryotes and plays an important role in cell proliferation,glycolipid metabolism,and melanin deposition.Recent studies have shown that it can activate the TGF beta signaling pathway.The transforming growth factor signaling pathway is closely related to the proliferation,migration and invasion of melanoma cells.However,the role of USF1 in melanoma is unknown.Objective: To investigate the expression level of USF1 in human melanoma cells and the effects of USF1 on the development of melanoma and its mechanism.Methods: The relative expression of USF1 in normal human melanocyte PIG1 cells and melanoma 1205-Lu,DO4,WM3211 and WM278 cells were processed and analyzed by qRT-PCR and Western blotting.The cell morphological changes in 1205-Lu cells after transfection with USF1 were observed by high-times optic microscope.Cell migration was determined using in vitro scratch test.In order to analyze the effects on epithelial-mesenchymal transition in 1205-Lu cells after transfection with USF1,the relative expression of E-cad,?-SMA,Vi and FN in 1205-Lu cells after transfection with USF1 were processed and analyzed by qRT-PCR and Western blotting.In order to further analysis the mechanism of USF1 caused the development of melanoma,the relative expression of TGF-?,Smad2 and ?-SMA in 1205-Lu cells after transfection with USF1 were processed and analyzed by qRT-PCR and Western blotting and the relative expression of TGF-?,Smad2 and ?-SMA in 1205-Lu cells after transfection with si-USF1 were processed and analyzed by qRT-PCR and Western blotting.Finally,the relative expression of Smad2,E-cad,?-SMA,Vi and FN in 1205-Lu cells after the single or combined transfection with si-USF1 and TGF-? were processed and analyzed by Western blotting.The cell morphological changes in 1205-Lu cells after the single or combined transfection with si-USF1 and TGF-? were observed by high-times optic microscope.Results: The relative m RNA and protein expression levels of USF1 were significantly enhanced in melanoma cell lines,1205-Lu,DO4,WM3211,and WM278,compared with normal human melanocytes PIG1 and the differences of the results were statistically significant(P<0.05).The USF1 expression levels in 1205-Lu cells was the highest.The 1205-Lu cells demonstrated an elongated and spindle-shaped morphology after transfection with USF1.The migration capacity in 1205-Lu cells after transfection with USF1 was significantly higher than that in blank control group.After transfection with USF1 48 h,the relative m RNA and protein expression levels of ?-SMA,Vi and FN in 1205-Lu cells were significantly higher than that in blank control group(P <0.05)and the relative m RNA and protein expression levels of E-cad were significantly lower than that in blank control group(P<0.05).The relative m RNA and protein expression levels of TGF-?,Smad2 and ?-SMA in 1205-Lu cells after transfection with USF1 48 h were significantly higher than that in blank control group(P <0.05)and the relative m RNA and protein expression levels of TGF-?,Smad2 and ?-SMA in 1205-Lu cells after transfection with si-USF1 48 h were significantly lower than that in blank control group(P<0.05).After 1205-Lu cells intervened by USF1 si RNA,the relative protein expression levels of Smad2,?-SMA and FN were significantly lower than that in blank control group(P<0.05).The relative protein expression levels of Smad2,?-SMA,Vi and FN in 1205-Lu cells after transfection with TGF? 48 h were significantly higher than that in blank control group and combined transfection with si-USF1 and TGF? group(P<0.05).Meanwhile,the relative protein expression levels of Smad2,E-cad,?-SMA,Vi and FN were no statistically significant between blank control group and combined transfection with si-USF1 and TGF? group(P>0.05).The 1205-Lu cells demonstrated an elongated and spindle-shaped morphology after transfection with TGF? and TGF-?-induced cell morphological changes could be partially reversed by knockdown of USF1.Conclusion: USF1 was high expression in human melanoma cells and overexpression of USF1 prompts the epithelial-mesenchymal transition process through the accumulation of TGF-?1production in melanoma cells.USF1 may be a novel therapeutic target of melanoma.