Hey1 Promotes Migration And Invasion Of Melanoma Cells Via UpregΜlating GRB2 To Activate PI3K/AKT Signalling

Objective: Hes related family b HLH transcription factor containing YRPW motif1(Hey1)is a target gene of Notch signaling pathway.Increasing evidence indicates that Notch signaling regulates multiple cell biological processes in malignant melanoma.As a target gene of Notch signaling,Hey1 is a wide-ranging transcription factors,Hey1 plays an important role in the development and progression of multiple tumors,but its role and underlying mechanisms in melanoma cells were neglected.The objective of this study was to investigate the effect of Hey1 on the biological function of melanoma cells and to elucidate the underlying mechanisms.Methods: We used bioinformatics tools,immunohistochemistry,and western blot analysis to evaluate the expression of Hey1 in melanoma tissues and cell lines.Both in vivo and in vitro xenograft models were used to determine the effect of Hey1 on the malignant behaviour of the melanoma cell line A375.High-throughput sequencing was performed on A375 after interference with Hey1 to detect differentially expressed genes,and KEGG enrichment analysis was used to analyze the signaling pathways involved in the differentially expressed genes.Immunofluorescence was used to detect the co-expression of Hey1 and GRB2 in melanoma tissues.Western blot was used to detect the effects of Hey1 on EMT related proteins,matrix metalloproteinases(MMPs)and key signaling molecules in/PI3 K / Akt signaling pathway.Results: Hey1 was markedly upregulated in melanoma tissues and cell lines.In vitro,interference with Hey1 significantly inhibited the migration and invasion of melanoma cells,increased the expression of E-cadherin,decreased the expressions of N-cadherin,vimentin and snail,and the expression of matrix metalloproteinases MMP2,MMP7 and MMP9 are also decreased.However,the results of that in the forced expression group were contrary to the interference group.In a xenograft animal model,the tumor volume of the Hey1 interfered group was significantly reduced and none of lung metastasis cell islands were detected,whereas the tumor volume and number of lung metastases in the forced expression group were increased,compared to the control groups respectively.Finally,232 coding genes were identified by high-throughput sequencing.Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that GRB2/PI3K/AKT signalling changed significantly.Western blot analysis showed that AKT and PI3 K expressions was not significantly changed;however,GRB2,p-PI3 K,p-AKT(ser308),and p-P21 expressions was significantly decreased in the interference group,whereas the trend of protein expression in forced expression group was opposite to that of interference group.Conclusions: Hey1 promotes the invasion and metastasis of melanoma,but its role may not be confined to the current research results.In addition,Hey1 is an effector of the NOTCH signalling pathway,but this study shows that Hey1 can promote the expression of key signaling molecules in the process of Grb2 / PI3 K / Akt signal activation.Thus,we speculate that Hey1 may play an important role in the crosstalk between the Notch and other signalling pathways.