Expression Of RGS4 And Its Role In Regulation And Development Of Malignant Melanoma

BackgroundMelanoma is a highly malignant tumor derived from cutaneous melanocytes.It has high metastasis rate and poor prognosis.Among them,high aggression,drug-resistance,early blood and lymphatic metastasis are the primary causes of melanoma death.The G-protein-coupled receptors(GPCRs)are the largest cell surface proteins family in eukaryotic organisms and have most diverse group of membrane receptors.Signal transduction via GPCRs is the basis of most physiological processes.In addition,GPCRs activation or expression abnormalities are intimately associated with cancer cell proliferation,tumor growth and metastasis.The Regulator of G-protein Signaling(RGS)proteins are initially characterized as inhibitors of signal transduction cascades induced by GPCRs due to their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins.Recent studies have revealed that many RGSs were involved in tumor development.The regulator of G protein signaling 4(RGS4)is a member of the B/R4 group.It works as a multifunctional scaffold protein for assembling proteins involved in regulatory signal transduction.Therefore,it’s very necessary to study the function and molecular mechanism of RGS4 for the diagnosis of melanoma.In view of the correlation between RGS4 and cancer,we have investigated the expression of RGS4 in melanoma,and found that the expression level of RGS4 is low in melanoma tissues,compared with the melanocytic nevus tissues as normal control.The specific role and mechanism of melanoma is not clear,we need further study.However,the physiological function of RGS4 in malignant melanoma is not well known so far.ObjectiveTo investigate the role of RGS4 in melanoma progression,we examined the expression of RGS4 in melanoma tissues and melanocytic nevus tissues by immunohistochemical(IHC)staining,and analyzed the correlations of RGS4 expression with clinicopathological characteristics of melanoma patients.In addition,we also examined the expression of RGS4 in melanoma cells and melanocytic cells by western blot.By manipulating RGS4 expression in melanoma cells,we determined the role of RGS4 in regulating melanoma cell proliferation,migration and invasion in vitro,and clarified the potential mechanisms underlying tumor-suppressor activity by RGS4 in melanoma.Methods1.Immunohistochemistry was used to detect the expression level of RGS4 in melanoma tissues and melanocytic nevus tissues.Analysis the correlation of RGS4 and clinical pathological characterstics in melanoma.2.Western blot was used to test the expression levels of RGS4 in human epidermal melanocyte cell line(HEM)and melanoma cell lines(M14,A375,UACC62,UACC257).RGS4 vector was formed using human full length RGS4 cDNA linked with the pcDNA3.1 vector to induce RGS4-overexpression in melanoma cells.Small interfering RNA was used to induced RGS4-downregulation in melanoma cells.3.CCK8 and colony formation assays were used to measure the influence of RGS4 on cell proliferation in vitro;TUNEL assays was tested to determine the effect of RGS4 on cell apoptosis in vitro;western blot assays were used to examine the expression levels of apoptosis-related proteins(Bcl-2.Bax.Caspase3.Cleaved caspase3)after manipulating RGS4 expression.4.Transwell assays were performed to investigate RGS4 effect on the migration and invasion of cancer cells in vitro;Western blot assays were used to examine the expression of biomarkers for EMT(E-cadherin,N-cadherin)and matrix metallopr oteinases(MMP2,MMP9)after manipulating RGS4 expression.5.Western blot was used to detect the phosphorylation of AKT,Cyclin D1 and E2F1,after manipulating RGS4 expression.PI3K inhibitor LY294002 was applied to corroborate the effect of PI3K/AKT pathway in RGS4-mediated tumor malignant behavior.Results1.RGS4 expression is remarkably decreased in melanoma tissues and cells.There were strong immunohistochemistry staining of RGS4 in melanocytic nevus tissues while it significantly decreased in melanoma tissues.RGS4-high expression rate(75%,6/8)in melanocytic nevus tissues was obviously higher than the rate(27.5%,11/40)in melanoma tissues(P<0.05).Meanwhile,we found that RGS4 expression was significantly correlated with TNM stage(P<0.01).In addition,we examined the expression of RGS4 in melanoma cell lines(M14,A375,UACC62,UACC257)and in normal skin cell line(Human Epidermal Melanocytes,HEM)as control by using western blot.Our results showed that RGS4 expression level was much lower in melanoma cells than in HEM cells2.RGS4 significantly inhibits melanoma cell proliferation in vitro.To investigate the biological functions of RGS4 in melanoma development,we selected A375 cell,with endogenous low RGS4 expression,to be transfected with pcDNA3.1-RGS4.The pcDNA3.1-RGS4 vector increased expression of RGS4 efficiently.CCK8 assays and colony formation assays confirmed the ability of RGS4 to suppress A375 cell proliferation.In addition,the expression of antiapoptotic protein Bcl-2 decreased,while the apoptotic protein Bax increased in pcDNA3.1-RGS4 transfected cells compared with the positive control.A375 cell with low endogenous RGS4 expression was selected for the transfer of siRGS4.The siRGS4 decreased expression of RGS4 efficiently.As CCK8 and colony formation assays showed,low level of RGS4 significantly promoted A375 cell viability.TUNEL assays confirmed that the apoptosis rate was significantly decreased compared with control cells.Meanwhile,western blot results showed that,the expression of antiapoptotic protein Bcl-2 was increased,while the apoptotic protein Bax and Cleaved caspase3 decreased in siRGS4-transfected cells.3.RGS4 inhibits melanoma cell migration and invasion in vitro.Trans-well assays ± Matrigel demonstrated that A375 cell transfected with pcDNA3.1-RGS4 vector had lower invasive activity than control cells.Western blot results showed that the protein levels of MMP2 and MMP9 were notably down-regulated with overexpression of RGS4.Up-regulation of E-cadherin was also observed in RGS4 overexpression A375 cells.Meanwhile,A375 cell transfected with siRGS4 had higher invasive activity than control cells.Western blot results showed that the protein levels of MMP2 and MMP9 were notably up-regulated with low-expression of RGS4.Down-regulation of E-cadherin and up-regulation of N-cadherin were also observed in RGS4 knockdown A375 cell.4.RGS4 inhibits E2F1 and Cyclin D1 via PI3K/AKT pathway.Western blot results showed that the expression of p-Akt was decreased in stable pcDNA3.1-RGS4 transfected A375 cell line.Meanwhile,RGS4 knockdown markedly stimulated the expression of p-Akt compared to the nagitive control in A375 cell line.Our data indicated that the expression Cyclin D1 and E2F1 was decreased in stable pcDNA3.1-RGS4 transfected A375 cells and increased in stable siRGS4 transfected A375 cells.LY294002 treatment further inhibited the expression of p-Akt,Cyclin D1 and E2F1,while knockdown of RGS4 markedly enhance the effect of LY294002 treatment.These results confirmed that RGS4 inhibited the expression of E2F1 and Cyclin D1 through inactivation of PI3K/Akt pathway.5.Knockdown of RGS4 significantly promotes cell proliferation,migration and invasion in M14 cell line.To further confirm the effect of knockdown of RGS4 on melanoma,we selected M14 cell line for cell function assays.Colony formation assays results showed,M14 cell viability was significantly promoted by downregulation of RGS4.Trans-well assays ±Matrigel demonstrated that M14 cell transfected with siRGS4 had higher invasive activity.The absent of RGS4 notably up-regulated the protein levels of MMP2 and MMP9.Meanwhile,the expression of anti-apoptotic protein Bcl-2 increased,while the apoptotic protein Bax decreased in siRGS4-transfected cells.Conclusion1.For the first time,our studies found that the expression of RGS4 is downregulated in melanoma tissues and cells.Furthermore,RGS4 expression and TNM stage were interrelated in clinic pathological factors by immunohistochemistry.2.RGS4 inhibits melanoma cell proliferation,migration and invasion in vitro.3.RGS4 inhibited melanoma cell proliferation and migration due to the inactivation of CyclinD1 and E2F1 via GPCR-mediated PI3K/AKT pathway.RGS4 might be a new therapeutic target for malignant melanoma.