| Background Melanoma,a malignancy deriving from melanocytes,has the highest mortality among skin cancers.Although the molecular targeted therapy for gene mutation or tumor immunotherapy provides a new treatment for melanoma patients,the side effects and drug resistance have greatly reduced the treatment efficiency and clinical application.The results suggest that there are other regulatory mechanisms in the development of melanoma.The study of the unknown mechanism will help to improve the level of diagnosis and treatment of melanoma.Autophagy is an important physiological process in the cell,through the degradation of degeneration,damage and loss of function of organelles and proteins to maintain homeostasis within the cell and provide energy supply necessary for cells under stress conditions.Recent studies showed that autophagy is important for maintaining tumor growth can inhibit tumorigenesis,with early and late dual role in promoting tumor progression in melanoma,but the upstream regulatory mechanism mediated by this dual role has not been fully elucidated.As a member of the Sirtuins family,SIRT6 plays an important role in DNA repair,telomere length maintenance,gene expression,metabolism,aging,and inhibition of inflammation.At present,it is considered to be the major regulator of intracellular glucose homeostasis,which can affect the metabolism through the growth of a number of network regulatory mechanisms,is a key regulator of tumor energy metabolism.The results showed that the level of autophagy mediated by SIRT6 could play an important role in promoting oxidative damage induced by oxidative stress and smoking induced apoptosis of bronchial epithelial cells.This phenomenon suggests that SIRT6 may be one of the reasons for the differential expression of autophagy in melanoma.However,these studies have focused on the non neoplastic diseases such as aging,inflammation,and so on.The regulation and mechanism of autophagy in SIRT6,especially in melanoma,is unclear.Further research on this problem will help to deepen the understanding of SIRT6 regulation of autophagy,and expand the spectrum of the two genes involved in regulation.It can provide new ideas and therapeutic targets for the clinical diagnosis and treatment of melanoma.Based on the above findings,we propose the following hypothesis: in different stages of melanoma,SIRT6 through autophagy regulation of intracellular inhibition of early melanoma growth and stimulate the growth of advanced melanoma play.Objectives 1.To analyse and verify the correlation between SIRT6 expression and Autophagy level in melanoma;2.To verify the regulation fuction of SIRT6 on autophagy and the biological roles.3.To discuss the molecular mechanism of autophagy mediated by SIRT6.Methods 1.q RT-PCR and Western blot was performed to testify the m RNA and protein expression of SIRT6 in benign nevus and different stages of melanoma or melanocytes and melanoma cells;immunofluorescence to testify the position of SIRT6 and LC3 in cells;The correlation between SIRT6 expression and autophagy was analyzed;2.CCK8 assay and flow cytometry analysis revealed the proliferation and cell cycle distribution with SIRT6 overexpression/knockdown;Western blot and flow cytometry were performed to testify the effect of SIRT6 to apoptosis in melanoma cells treated with SIRT6 overexpression/knockdown;3.CCK8 assay and flow cytometry analysis revealed the proliferation and cell cycle distribution with ATG5 overexpression/knockdown or autophagy regulator(choloroquine or rapamycin);Western blot and flow cytometry were performed to testify the effect of ATG5 or autophagy regulator to apoptosis in melanoma cells;4.Western blot,immunofluorescence and TEM were performed to testify the effect of SIRT6 on autophagy in melanoma cells treated with chloroquine after SIRT6 overexpression or knockdown;5.CCK8 assay and flow cytometry analysis revealed the proliferation and cell cycle distribution with SIRT6 overexpression/knockdown after treated with autophagy regulators(choloroquine or rapamycin);Western blot and flow cytometry were performed to testify the effect of autophagy regulator to apoptosis in melanoma cells with SIRT6 overexpression/knockdown;6.q RT-PCR was performed to testify the m RNA expression of IGF1 R in benign nevus and different stages of melanoma or melanocytes and melanoma cells;Western blot was performed to testify the activation og IGF-Akt signaling related protein;The correlation between SIRT6 expression and IGF-Akt was analyzed;7.Western blot was performed to testify the effect of SIRT6 to the activation of IGF-Akt signaling,the total and phosphorylated protein expression were detected in melanoma cells after Akt overexpression or inhibition with SIRT6 overexpression/knockdown;8.Western blot was performed to testify the effect of the IGF-Akt signaling to autophagy,the total and phosphorylated protein expression were detected in melanoma cells after Akt overexpression or inhibition with SIRT6 overexpression/knockdown;9.CCK8 assay and flow cytometry analysis revealed the proliferation and cell cycle distribution with SIRT6 overexpression/knockdown after treatment with Akt overexpression or inhibition;flow cytometry were performed to testify its effect to apoptosis in melanoma cells with;10.Xenograft model by subcutaneous implantation of SIRT6 overexpressed primary melanoma cells were employed to confirm our findings in vivo.The volume and weight of the tumor from subcutaneous implantation,the metastatic ablity of subcutaneous implantation were detected.Results 1.Both m RNA and protein levels of SIRT6 were significantly down-regulated in primary melanomas(PM),but up-regulated in metastatic melanomas;the expression of SIRT6 was in a prominent positive correlation with LC3 and a significant negative correlation with p62;2.The result of CCK8 assay and flow cytometry demonstrated that inhibition of cell proliferation and cell-cycle arrest in G1 phase in both primary melanoma cells with SIRT6 overexpression and metastatic melanoma cells with SIRT6 silence.Moreover,we found that SIRT6 could promote apoptosis of primary melanoma cells but impeded apoptosis of metastatic melanoma cells;3.CCK8 assay and flow cytometry analysis revealed that overexpressed ATG5 or rapamycin treatment resulted in a significant inhibition of cell proliferation and G1 phase cell-cycle arrest in primary melanoma cells.On the other hand,ATG5 knockdown or choloroquine reduced the proliferation and arrest cell-cycle in G1 phase in metastatic melanoma cells.Moreover,ATG5 promoted apoptosis in primary melanoma cells but suppressed apoptosis in metastatic melanoma cells.Subsequent immunoblotting analysis also showed the consistent alterations of the anti-apoptotic protein Survivin,the pro-apoptotic proteins Bax and cleaved PAPR-1 with ATG5 intervention in different melanoma cell lines;4.The result of western blot,transmission electron microscopy and immunofluorescence demonstrated that autophagy was induced/inhibited in melanoma cells after SIRT6 overexpression/knockdown;5.CCK8 assay and flow cytometry analysis revealed the inhibition of autophagy by chloroquine partially rescued SIRT6 overexpression induced cell proliferation prevention,cell-cycle arrest and cell apoptosis.On the other hand,autophagy activation by rapamycin also incompletely reversed the impacts of SIRT6 knockdown on metastatic melanoma cells;6.The result of IGF1 R m RNA and protein level was significantly increased in melanomas at different stages compared with benign nevus and normal melanocytes.the expression of SIRT6 was in a prominent negative correlation with p-IGF1 R and p-Akt Ser473;7.Western blot demonstrated that IGF-Akt related prtoein were all prominently phosphorylated in both melanoma tissues and cell lines,but the phosphorylation of these IGF-Akt pathway molecules were slightly dampened in metastatic melanoma compared with primary melanoma after Akt overexpression or inhibition with SIRT6 overexpression/knockdown;8.Western blot showed that autophagy level was impaired in melanoma cells treated with Akt overexpression or inhibition combined with SIRT6 overexpression/knockdown;9.CCK8 assay and flow cytometry analysis revealed Akt overexpression ameliorated the inhibition of cell viability and cell-cycle progression in SIRT6 overexpressed primary melanoma cells.In tandem,Akt inhibitor resulted in increased cell viability and cell-cycle progression in SIRT6 silenced metastatic melanoma cells,and the numbers of apoptotic cells was significantly reversed by Akt intervention in these melanoma cell lines accordingly 10.Xenograft model by subcutaneous implantation showed that compared with the indicated control groups,the tumor size and weight were significantly decreased in mice implanted with SIRT6 overexpressed primary melanoma cells and SIRT6 silenced metastatic melanoma cells.Mice received implantation of SIRT6 silenced metastatic A2058 cells possessed less liver metastases through gross inspection and pathological analysis.In addition,phosphorylation of IGF1 R,Akt and downstream molecules decreased in xenografts implanted with SIRT6 overexpressed primary melanoma cells.Nevertheless,in xenografts implanted with SIRT6 silenced metastatic melanoma cells,the phosphorylated IGF1 R,Akt,m TOR and p70S6 K expression significantly increased.The expression of the autophagy level showed a contrary trend towards the change of IGF-Akt pathway.Conclusion In conclusion,we demonstrated that SIRT6 bidirectional expression pattern in the early and late stages of melanoma,and it can promote autophagy through negatively regulating IGF-Akt signaling,which involved in the regulation of melanoma cell growth.Our findings deepen our understanding of the regulation of autophagy by SIRT6 and expand the spectrum of the two genes related diseases.To our knowledge,the role of SIRT6 in facilitating cell-protective autophagy in melanoma has not been reported previously,whcih provide new ideas and therapeutic targets for the clinical diagnosis and treatment of melanoma. |
PDF Full Text Request