Rsf-1 Regulates Cell Proliferation,Invasion,Apoptosis And Chemoresistance Through NF-?B Signaling In Melanoma

Objective: Melanoma,also named MM,malignant melanoma,is one kind of highly malignant tumor that develops from the pigment-containing cells known as melanocytes.Melanomas always occur in human skin but may rarely appear at intestines,mouth or eye.In recent years,there is an increasing tend for its mortality and also as morbidity.Its etiology is not very clear yet and the first important cause of melanoma is always thought to be exposed in much ultraviolet light(UV)in those who has low skin pigment levels.So,to prevent melanoma,there are many methods,such as using umbrella to avoid UV light,take glasses to avoid sunscreen.To treat MM,the most ordinary method is mainly surgery removal and some patients can be cured when MM has not be spread yet.In those who has already got its spread,some methods such as biologic therapy,immuno-therapy and also chemotherapy,even radiation therapy can bring some advantage for the survival.But,for early metastasis and the poor sensitivity to the chemotherapy always results in most melanoma patients poor prognosis.Therefore,it is urgent to identify molecular mechanisms of melanoma and find new therapeutic targets and agents to improve the prognosis of melanoma.Among many kinds of complex factors which are involved in tumorgenesis,gene amplification is a common mechanism underlying oncogenic activation in human cancer and the 11q13.5 region is well known to be amplified in many types of malignant diseases including ovarian,breast,and other part of human body.The gene on 11q13.5 amplification has been proposed to contain a number of potential candidate drivers,such as EMSY?Rsf-1 and so on.Rsf-1,(also known as HBXAPa)showed the highest correlation between DNA copy number and RNA copy number in ovarian cancer tissues.Some researchers gathered evidence that Rsf-1 has played much important role in the selection or development of the 11q13 amplicon in 13– 17% of ovarian cancers at the DNA,RNA,protein,and functional levels.Importantly,elevated levels of Rsf-1 correlated with poor prognosis while depletion of Rsf-1 could reduce the proliferative ability of cells of ovarian cancer,playing an major role of Rsf-1 amplification in maintaining cell survival and growth in ovarian cancer.In breast cancer,we also can observe overexpression of Rsf-1,for a less extent.Most recent study showed overexpression of Rsf-1 was accompanied by overexpression of hSNF2 H and the role of Rsf-1 in prompting cell proliferation was dependent on the formation of the Rsf-1/hSNF2 H complex.Previous study using interaction network analysis showed that Rsf-1 expression was associated with changes in the expression of some genes that involved in NF-? B and other pathways.The protein expression of Rsf-1 in melanoma has not yet been examined.In addition,the biological roles and molecular mechanisms of Rsf-1 in melanoma cells are still unclear.In order to address the above questions,we examined Rsf-1 expression in melanoma tissues by immunohistochemistry.Then we also investigated the association of Rsf-1 with proliferative,invasive and apoptotic abilities in melanoma cell lines,and explored the underlying molecular mechanism.Methods and results: 1.We identified Rsf-1 expression pattern and compared the difference of Rsf-1 expression between melanoma tissue samples and normal skin tissues.In this study,we examined Rsf-1 expression in 52 cases of melanoma tissue by immunohistochemistry.Normal normal skin tissues showed weak or negative staining.In 40.4%(21/52)of melanoma tissues,we found Rsf-1 overexpression,which was located in the nucleus of the tumor cells.2.We down/up-regulated Rsf-1 to detecte the biological roles of Rsf-1 on melanoma cell growth and invasion.We examined Rsf-1 expression in MV3,M14 and A375 cell lines and found strong Rsf-1 expression in MV3 and A375 cells.Then we employed Rsf-1 plasmid in M14 cells,Rsf-1-targeting siRNAs in MV3 and A375 cell lines.We use MTT method and colony formation assays showed Rsf-1 knockdown inhibited cell growth situation and colony formation.Cell cycle was examinde by fluorescence activated cell sorting(FACS),and also it was demonstrated that Rsf-1 depletion has made some decrease for S phase percentage and increase G2 phase percentage.And,matrigel invasion assays showed that Rsf-1 downregulation suppressed cell invasion.3.In order to investigate the mechanism underlying melanoma progression induced by Rsf-1,we examined the effect of Rsf-1 on cell proliferation and invasion related molecules.We also examined the Rsf-1 effect on cyclin E,MMP2 and p-I?B expression.It was revealed by Western blotting Rsf-1 depletion decreased MMP2,cyclin E expression and I?B phosphorylation.Immunoprecipitation showed that Rsf-1 could interact with hSNF2 H in both melanoma cell lines.4.We down/up-regulated Rsf-1 to detecte the biological roles of Rsf-1 on melanoma cell apoptosis,survival and mitochondrial membrane potential after being treatment by paclitaxel.We also investigated the effect of Rsf-1 depletion on cell apoptosis after 5uM paclitaxel treatment.MTT showed that Rsf-1 knockdown could increase cell apoptosis and decrease cell survival.JC-1 assay showed that Rsf-1 knockdown decreased mitochondrial membrane potential in melanoma cells.5.In order to investigate the mechanism underlying melanoma apoptosis inhibited by Rsf-1,we examined the effect of Rsf-1 on cell apoptosis related molecules.we examined the effect of Rsf-1 on Bcl-2,cIAP1,Bax and cIAP2 expression.It was revealed by Western blotting analysis that Rsf-1 depletion could decrease the level of Bcl-2,cIAP1,cIAP2,while increase the expression of Bax.Conclusion:1.Rsf-1 expression in melanoma tissue is much more higher.The positive rate of Rsf-1 expression in melanoma tissue is 40.4%(21/52).2.Rsf-1 depletion inhibited cell proliferation and invasion in melanoma.3.Expression of cyclin E and MMP2 can be decreased by Rsf-1 knockdown cells,inhibited I?B phosphorylation in melanoma cells.4.Rsf-1 could interact with hSNF2 H in melanoma cell lines.5?Rsf-1 knockdown could increase apoptosis of cells,decrease survival of cells and mitochondrial membrane potential in melanoma cell line.6?Rsf-1 depletion could decrease the level of Bcl-2,cIAP1,cIAP2,while increase the expression of Bax.7?Rsf-1 depletion could inhibit NF-?B signaling pathway in melanoma cells.