Effect Of AFF4 On Migration And Invasion Of Melanoma And Its Mechanism

Background and objective:The morbidity of melanoma is significantly high in recent decades.Clinical characteristics of human cutaneous melanoma include no particular symptoms in the early stage,easy invasion and metastasis.Once metastasis occurs,it is difficult to obtain satisfactory therapy in clinic.However,the mechanism for invasion and metastasis of melanoma is not still clear.Excitedly,the treatment of melanoma invasion and metastasis according to research on the perspective of EMT is one new strategy.AFF4,a transcriptional regulation factor,involves in cell proliferation and cell different process.Previous studies indicate that AFF4 is associated with regulation of tumor development.However,few investigations have focused on the expression and function of AFF4 in melanoma.Firstly,this study investigated the AFF4 expression and development in melanoma,and analyzed the effects of AFF4 expression based on the clinicopahtological and survival dates.Secondly,the relationships of AFF4 with melanoma cell proliferation,invasion and migration were studied via molecular biological experiments in vivo and in vitro.Finally,we discussed the downstream gene mediated by AFF4 and its potential mechanism in melanoma.The aim of this study is to clarify the role of AFF4 in the development of melanoma and its underlying downstream mechanisms,so as to provide theoretical basis and candidate targets for targeted therapy of melanoma.Methods:For the above purposes,we adopted the following research methods1.Study on the expression of AFF4 in melanoma patientsA bioinformatics analysis was made to evaluate the expression of AFF4 genes based on the mRNA microarray data.We analyzed the expression of AFF4 in primary melanoma and metastatic melanoma using bioinformatics database GEO profile.Based on this,the immunohistochemistry was applied to analyze the expression of AFF4 in 117 cases of melanoma and 68 cases of nevi,so as to explore the relationship between AFF4 gene and clinical pathological characteristics of melanoma.Finally,the correlation between AFF4 gene and prognosis would be analyzed based on survival data.2.The effect of AFF4 gene on invasion and metastasis of melanomaThe interference technique using lentivirus-mediated RNA was applied to silence AFF4 and transfect AFF4 overexpression plasmid with the purpose of elucidating the biological function of AFF4 in A375 and A2058 cell lines.The effects of AFF4 on the proliferation of melanoma were explored by CCK8 method.Then,the transwell invasion and scratch test were adopted to detect the effect of AFF4 on the invasion and migration of melanoma cells.Furthermore,western blotting was used to detect the changes of EMT markers after knock-down and overexpression of AFF4.3.Potential mechanism of AFF4 regulating c-jun signaling pathway mediating EMT to affect invasion and metastases of melanomaWestern blotting was applied to detect the expression of c-jun and phosphorylated c-jun after knockdown and overexpression of AFF4.To verify whether AFF4-mediated EMT could affect invasion and metastasis of melanoma by regulating c-jun/MMP2 signal axis,the siRNA and control series of c-jun were constructed,and rescue experiment was implemented to verify whether knockdown of c-jun could reverse the influence of AFF4 on melanoma invasion and metastasis Finally,the effect of c-jun gene knockdown on EMT of melanoma cells induced by AFF4 overexpression was detected via western blotting.4.Effect of AFF4 on the tumorigenicity of melanoma in vivoTwo groups of A375 cells(shAFF4 and scramble groups)were cultured and then injected subcutaneously into the flank of BALB/c-nu node mice(6 for each group).The general physical state,tumor formation rate and tumor growth of nude mice were routinely.observed and measured in the first 31 days.Meanwhile,the tumor volumes and weights of mice were measured,and the IHC analyses were implemented to analyze the AFF4,c-jun and Ki-67 expression in xenografts.Results:1.Study on the expression of AFF4 in melanoma patientsThe GEO profile database showed that the AFF4 mRNA expression in metastatic melanoma tissues(52 cases)was significantly higher than that in primary cutaneous melanoma tissues(31 cases)(P=0.0214)(GEO profile ID:74500763).Furthermore,AFF4 protein expression in the cutaneous melanoma(72.15%)and mucosal melanoma samples(65.5%)was significantly higher than that in the benign nive tissue samples(45.59%)(P=0.001 and 0.019,respectively).There was no significant difference in AFF4 expression between cutaneous melanoma and mucosal melanoma(P=0.310).In addition,there was no significant difference in AFF4 expression between acral melanoma(33/47)and non-acral melanoma(18/32)(P=0.151).However,the high AFF4 protein expression was remarkably correlated with Clark level(P=0.023),AJCC stage(P=0.011),Breslow thick(P=0.032).There was no significantly different between the expression of AFF4 and patient's age,gender,ulcer status,mitotic rate,tumor infiltrating lymphocyte(TIL)and Ki-67 expression(P>0.05).Furthermore,Kaplan-Meier analyses revealed that the median survival of AFF4 high expression group was 50 months,and the median survival time of AFF4 low expression group was 74 months,the difference was significant(P=0.032).Overall survival(OS)were worse for the patients with high AFF4 expression than for the patients with low expression.The AFF4 protein expression is closely related to the prognosis of patients with cutaneous melanoma.2.The effect of AFF4 gene on invasion and metastasis of melanomaMelanoma cell models was successfully constructed via knockdown and overexpression of AFF4.For A2058 and A375 cells,the proliferation,invasion and migration ability of melanoma cell lines with silencing AFF4 were reduced,while the melanoma cell lines after transfected overexpression plasmid had the opposite result.Overexpression AFF4 can reverse the effect of knockdown AFF4 on the biological function of A375 cells by rescue assay.Moreover,western blotting analysis showed that E-cadherin was elevated,while N-cadherin and Vimentin was decreased by knockdown AFF4 expression.Similarly,overexpression AFF4 caused down-regulated expression of E-cadherin as well as up-regulated expression of N-cadherin and Vimentin.3.Potential mechanism of AFF4 regulating c-jun signaling pathway mediating EMT to affect invasion and metastases of melanomaWestern blotting analysis of the expression of c-jun and p-c-jun were detected after AFF4 knockdown and overexpression in melanoma.The c-jun,p-c-jun(ser63)and p-c-jun(ser73)protein expression decreased after silencing AFF4 gene.On the contrary,the c-jun and p-c-jun(ser63),p-c-jun(ser73)protein expression increased due to AFF4 overexpression,which deduced that AFF4 could regulate c-jun signaling pathway.The AFF4-Vector-A375+si-c-jun can return the effect of AFF4 overexpression on invasion and metastasis by rescue assay.Moreover,Western blotting results showed that the expression of c-jun,p-c-jun(ser63),p-c-jun(ser73),MMP2,Vimtern and N-cadherin were up-regulated after overexpression AFF4,while the expression of E-cadherin was down-regulated.However,it had the opposite results in AFF4-Vector-si-c-jun A3 75 cell.Further,the expression of AFF4 was positively correlated with that of c-jun in melanoma tissue by IHC.4.AFF4 promotes tumorigenesis of melanoma cell in vivoIn order to detect the xenografts formation ability,subcutaneously injection in nude mice was performed.Xenografts formed by shAFF4-A375 cells was much smaller in volume(84.34± 19.85 cm3 for shAFF4 group,189.5±51.94 cm3 for scramble group,P=0.0057)and heavier in weight compared with the scramble-A3 75 cell(0.2417±0.06882 g for shAFF4 group,0.5083±0.1463g for scramble group,P=0.016).After AFF4 knockdown,the c-jun expression and Ki-67 index decreased in vtvo.Conclusion:1.AFF4 gene in melanoma tissue was significantly increased by the nevi tissue,and the expression level was positively associated with cutaneous melanoma development,which could be a promising indicator to predict the prognosis of patients with cutaneous melanoma.2.AFF4 promotes the invasion and migration of melanoma cells by mediating the EMT transformation.3.AFF4 may regulate c-jun so as to promote the invasion and migration of melanoma cells.4.The c-jun was regulated by AFF4 gene to promote melanoma tumorigenesis in vivo.In summary,AFF4 could play as an aggressive factor of melanoma,and AFF4/c-jun axis could be a potential therapeutic target for melanoma patients.