Mechanism Of HDAC3 Promote Epithelial-Mesenchymal Transition(EMT)in Melanoma

Background and Objectives Melanoma is a very malignant tumor.Early metastasis and recurrence are the main causes of death.Prevention of metastasis is the key to reducing melanoma mortality.Epigenetic signaling pathways are involved in the development and progression of a variety of tumors,and histone acetyltransferase 3(HDAC3)is involved in the development of many types of tumors(cholangiocarcinoma,pancreatic cancer,colon cancer).However,the role of HDAC3 in the development of melanoma is not clear.Previous literature in the our laboratory demonstrated that HDAC3 is elevated in metastatic lymph node tissues of malignant melanoma,and that HDAC3 can promote the proliferation of malignant melanoma by deacetylating p53;Here we first applied the histone deacetylase inhibitor MS-275 and find that it can simultaneously reduce the protein levels of HDAC3 and FUT7.Then we applied c DNA plasmid to upregulate HDAC3 and verified that HDAC3 can increase epithelial mesenchymal transition(EMT)ability of malignant melanoma cells by affect FUT7 protein and m RNA levels;GATA3 regulates its transcriptional activity by binding to the FUT7 genome.Our study found that HDAC3 interacts with GATA3 directly or indirectly in melanoma cells and regulates GATA3 protein levels by affecting GATA3 acetylation levels without affecting m RNA level.Our results in this study indicate that HDAC3 can regulate the expression of FUT7 gene and protein levels by deacetylating GATA3 in malignant melanoma cells,thereby affecting the EMT ability of melanoma cells,making HDAC3 as a therapeutic target for melanoma and Provide a theoretical basis for the combination of HDAC inhibitors and glycosyltransferase inhibitors.Methods: 1.Human melanoma cells A375 P and murine melanoma cells B16 were treated with different concentrations(0,5,10,and 20 μM)of histone deacetylase inhibitor MS-275 for 48 hours.Detected the protein expression of HDAC3 and Fut7 by Western blot.2.Transfected human melanoma cell line A375 P and mouse melanoma cell line B16 with Lipo 2000 to transiently transfect Vector or HDAC3 c DNA to up-regulate HDAC3,and Real-time PCR and Western blot were performed to verify the transient transfection efficiency.The effect of up-regulation of HDAC3 on epithelial mesenchymal(EMT)-associated proteins(N-cadherin,E-cadherin,Vimentin,and Snai)in melanoma cells were examined by Western blot and immunofluorescence.3.FUT7 c DNA was transiently transfected into human melanoma cell line A375 P with Lipo 2000 to up-regulate FUT7.Real-time PCR and Western blot were used to verify the transient transfection efficiency.Real-time PCR,Western blot and immunofluorescence were used to detect the effection of melanoma cell epithelial mesenchymal(EMT)-associated proteins(N-cadherin,E-cadherin,Vimentin,and Snai).4.Transfected human melanoma cell line A375 P and mouse melanoma cell line B16 with Lipo 2000 to transiently transfect Vector or HDAC3 c DNA to up-regulate HDAC3,Real-time PCR,Western blot and immunofluorescence were used to detect effects of m RNA and protein levels in FUT7 and GATA3 via up-regulate HDAC3 in melanoma cells.5.A375 P cells were transfected with Vector or HDAC3 c DNA or Si FUT7 or HDAC3 c DNA and Si FUT7 by Lipo2000 in vitro,and the expression of EMT-related marker proteins N-cadherin,E-cadherin and Vimentin was detected by Western blot.6.collected proteins of four malignant melanoma cells,A375 P,C8161,SK-MEL28 and B16 separately.The interaction of HDAC3 and GATA3 in cells was detected by immunoprecipitation and Western blot.HDAC3 was transiently transfected with mouse or HDAC3 c DNA in vitro by Lipo 2000.The effect of up-regulation of HDAC3 on the acetylation level and the ubiquitination of GATA3 was detected by immunoprecipitation and Western blot.7.Human melanoma cells A375 P and murine melanoma cells B16 were treated with different concentrations(0,2.5,5,and 10 μM)of proteasome inhibitor MG-132 for 6 hours or applied 5μM MG-132 after transient transfection of HDAC3 c DNA.Detected the protein expression of GATA3 Western blot.Results: 1.The histone deacetylase inhibitor MS-275 can simultaneously reduce the protein expression of HDAC3 and FUT7 and in a concentration-dependent manner in melanoma cells.2.The expression levels of N-cadherin,Vimentin and Snail were increased in the HDAC3 c DNA transfection group,while the protein level expression of E-cadherin was decreased.3.The m RNA and protein levels of N-cadherin,Vimentin and Snail were increased in the FUT7 c DNA transfection group,while the m RNA and protein levels of E-cadherin were decreased.4.In the HDAC3 c DNA transfection group,the m RNA and protein levels of FUT7 were increased,the expression level of GATA3 m RNA was basically unchanged,however,the protein level expression of GATA3 was decreased.5.The expression levels of N-cadherin and Vimentin were increased in the HDAC3 c DNA transfection group,while the protein level of E-cadherin was decreased.The protein levels of N-cadherin and Vimentin were decreased in the Si FUT7 transfected group.while E-cadherin protein levels were elevated,whereas protein expression levels of N-cadherin,E-cadherin,and Vimentin were reversed in the HDAC3 c DNA and Si FUT7 double transfection treatment groups.6.GATA3 interacts with HDAC3 in four malignant melanoma cells,A375 P,C8161,SK-MEL28 and B16.Decreased acetylation level and increased ubiquitination level of GATA3 in HDAC3 c DNA transfection group.Conclusions: 1.HDAC3 can promote the epithelial-mesenchymal transition(EMT)ability of malignant melanoma cells.2.HDAC3 can increase the epithelial-mesenchymal transition(EMT)ability of malignant melanoma cells by affecting the expression of FUT7 m RNA and protein levels.3.In malignant melanoma cells,HDAC3 binds to GATA3 and reduces the protein level by deacetylating GATA3,thereby affecting the expression of FUT7.