The Expression And Function Of Sirtuin 4 And Sirtuin 5 In Melanoma

Objective: Silence regulatory proteins(Sirtuins,Sirts)are a protein family with multiple metabolic regulatory activities.The Sirtuins family contains 7 major member proteins,among which Sirt 4 and Sirt 5 pocesses multiple enzymatic properties.Recent studies have found that Sirt 4 and Sirt 5 are extensively involved in cell survival,aging and metabolism control,and play a vital role in many important biological proceses such as stress responses,inflammation,tumorigenesis and energy production.Besides,they have also been shown to be potential in treatment against cancer,diabetes mellitus,obesity,Parkinson and Alzheimer’s diseases.Melanoma is a commonly seen cutaneous cancer in clinic,however,relationship among melanoma and Sirt 4/5 proteins have been quite under-investigated.The mechanisms by which Sirt 4/5 proteins regulate the growth,development and metastasis of melanoma remain unclear.Therefore,more detailed investigations are required in order to better illustrate this specific subject.In this work,we explored the expression pattern of Sirt 4/5,as well as some of the basic roles that they take to influence human melanoma.Methods: Immuno-histo-chemistry staining of Sirt 4 and Sirt 5 proteins was performed on a tissue microarray containing 82 melanomas and 18 nevi.The clinical information of the patients involved in the microarray was collected.The relationship between Sirt 4/5 expression level and melanona malignancy was analyzed through oridinal logisitic regression.Human melanoma cell line HTB140,C8161,A2058,435 S,A375 and normal human melanocyte PIG1 were cultured with use of DMEM,1640,M254,DMEM/F12 medium with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics.All cell cultures were maintained in a 37 °C humidified incubator with 5% CO2 and air.Protein and mRNA expressions were examined by western blotting and real-time qPCR assays.Sirt 5 gene editing was performed with use of CRISPR/Cas9 technology(knock-out)and plasmid transefection(overexpression).The migratory ability of melanoma cells were tested via wound healing assay.The apoptosis and cell cycle distribution of melanoma cells were examined with use of flow-cytometry.The proliferation index was tested through MTS proliferation assay.Results: Both Sirt 4 and Sirt 5 proteins were expressed in higher amount in human melanoma compared to benign nevi.Sirt 5 was found to be positively-related to the malignancy of melanoma on the tissue microarray(P<0.05),whereas Sirt 4 was not(P>0.05).Baseline mRNA and protein expressions of both Sirt 4 and Sirt 5 in human melanoma and melanocyte cell lines were observed.Sirt 5 gene was knock-out in C8161 and A375 cell line as demonstrated by DNA sequencing,however,Sirt 5 protein expression was not completely eradicated.Overexpression of Sirt 5 protein was successful in both C8161 and A375 cell line as proved by western blotting.Sirt 5 overexpression promoted the proliferation and migration ability in C8161 and A375 cell line shown by MTS and wound healing assays.Besides,Sirt 5 overexpression caused elevated the percentage of S phase in C8161 and A375 cell lines as demonstrated by cell cycle distribution assay,whereas it only caused promoted apoptotic rate of cells in A375,not in C8161,cell line shown by apoptosis assay.Conclusion: Both Sirt 4 and Sirt 5 proteins are regularly expressed in human melanoma and melanocytes.Melanoma with higher malignancy appears to have higher expression of Sirt 5 protein,and overexpression of Sirt 5 in melanoma causes elevated cell proliferation and migration ability,which lead us to a conclusion that Sirt 5 may promote melanoma development via up-regulation of cellular proliferation and migration.