The Role And Molecular Mechanism Of TRIP In Metastasis Of Choroid Melanoma

Choroidal melanoma(CM)is the second most common form of malignant melanoma and the most prevalent type intraocular malignancy,accounting for～70%of uveal melanomas(UM).At present,CM treatment is primarily based upon radiotherapy or surgical excision or a combination of them.CM patients,however,often suffer from tumor metastasis to distant tissues including liver(90%),lung(24%),and bone(16%).The median survival of metastatic CM patients in less than 1 year.Owing to the high rates of metastasis and limited therapeutic responsiveness associated with these tumors,CM cases account for approximately 13%of all melanoma-related mortality.It is therefore vital that the molecular mechanisms governing CM onset and progression be better understood so that more efficacious treatment strategies can be developed for patients in need.The process of epithelial-mesenchymal transition(EMT)wherein epithelial cells undergo transdifferentiation and adopt a more motile mesenchymal phenotype is a key step in metastatic tumor progression.In recent years,EMT in other solid tumors besides cancer has not been widely reported may because they are non-epithelium origin such as UM,derived from normal uveal melanocyte.However,it is reported that EMT can be applied to judge aggressiveness of UMs molecularly as an aggressiveness marker in carcinomas.The evolutionarily conserved helix-loop-helix(HLH)transcription factor Twist-related protein 1(Twist1)is capable of promoting EMT progression owing to its ability to simultaneously suppress the expression of epithelial marker proteins(E-cadherin)while promoting the expression of mesenchymal marker proteins(vimentin and N-cadherin).Many solid tumors exhibit Twistl overexpression,with the degree of such overexpression correlating with a more aggressive tumor phenotype and with poorer patient survival outcomes.Effective control of Twistl expression therefore represents a potential approach to controlling tumor metastatic progression via suppressing the EMT process.Twistl can undergo post-translational ubiquitination and consequent degradation under the influence of the ubiquitin-proteasome system(UPS),thereby controlling the intracellular levels of this protein.TRIP is an E3 ubiquitin ligase that was first detected as an inhibitor of NF-κB capable of interacting with TRAF.TRIP contains two coiled-coil domains and a RING domain,with the latter being essential for its ubiquitin ligase activity.Previous work has demonstrated that TRIP is involved in the tumorigenesis of several types of cancer,including breast cancers lung adenocarcinoma and liver cancer.However,little is known about the role of TRIP in CM.In this study,we analyzed of RNA-seq data from UM patients in TCGA,and found that elevated TRIP expression was predictive of better overall survival(OS).In addition,TRIP was highly overexpressed in better-prognosis disomy 3(D3)UM compared to poor-prognosis monosomy 3(M3)UM.We also determined that TRIP can function as an E3 ubiquitin ligase for Twistl,promoting its K48-polyubiquitination and consequent degradation,thereby controlling EMT progression.Overexpression of TRIP was sufficient to impair CM cell EMT,proliferation,and invasion.As such,our findings suggest that TRIP is a novel tumor suppressor in CM and may represent a viable target for therapeutic intervention.Objectives:1.To clarify the effect of TRIP on the proliferation and migration of choroidal melanoma cells.2.To investigate the role of TRIP in EMT of choroidal melanoma cells.3.To underline the molecular mechanism of TRIP.Methods:1.To analyze the relationship between TRIP and prognosis of patients with choroidal melanoma using the TCGA database2.Effect of TRIP on proliferation and metastasis of choroid melanoma cells2.1 Effect of TRIP overexpression on proliferation and metastasis of choroidal melanoma cells.TRIP-overexpressed plasmids and blank vectors were transfected into M619 or OCM-1 cells,respectively.24h later,TRIP expression was detected by Western blot.MTT assay was used to detect the effect of TRIP overexpression on the proliferation of choroidal melanoma cells.The effect of TRIP overexpression on the metastasis ability of choroidal melanoma cells was detected by Transwell assay.2.2 Effect of TRIP knockdown on proliferation and metastasis of choroid melanoma cells.TRIP specific siRNA were designed,and Ctrl siRNA and TRIP siRNA were transfected into M619 or OCM-1 cells,respectively.After 48h,Western blot detection of TRIP expression was performed to determine the interference efficiency.MTT assay was used to detect the effect of TRIP knockdown on the proliferation of choroidal melanoma cells.The effect of TRIP knockdown on the metastasis ability of choroidal melanoma cells was detected by Transwell assay.3.Effect of TRIP on EMT of choroidal melanoma cells3.1 Effect of TRIP overexpression on EMT of choroidal melanoma cells.TRIP overexpressed plasmid and blank vector were transfected into M619 or OCM-1 cells,respectively.After 24h,mRNA and protein expression levels of EMT-related molecular markers(E-cadherin,N-cadherin,and Vimentin)were detected by RT-PCR and Western blot.3.2 Effect of TRIP knockdown on EMT of choroidal melanoma cells.The control siRNA and TRIP siRNA were transfected into M619 or OCM-1 cells,respectively.After 48h,mRNA and protein expression levels of EMT-related molecular markers(E-cadherin,N-cadherin,and Vimentin)were detected by rRT-PCR and Western blot.4.The molecular mechanism of TRIP in the EMT process of choroid melanoma cells4.1 Influence of TRIP on EMT activating transcription factors expression.TRIP overexpressed plasmid and blank vector were transfected into M619 cells,respectively,expression levels of EMT activating transcription factors(Snail,Slug,ZEB1,Twistl)were detected by Western blot.4.2 Interaction of TRIP and EMTactivating transcription factors.4.2.1 Flag-TRIP and Myc-Snail,Slug,ZEB1 and Twistl plasmids were co-transfected into HEK293T cells,respectively.Co-immunoprecipitation technology was used to detect the combination of TRIP and these transcription factors.4.2.2 The interaction of endogenous TRIP and the target molecules found in above experiments was detected in M619 cells.4.2.3 HEK293T cells were respectively transfected with different labels of blank vectors,TRIP WT plasmids and TRIP△C51 plasmids lacking of the RING domain,co-immunoprecipitation technology was used to detect the binding sites of TRIP and its target molecules.4.3 Effect of TRIP on the ubiquitination of its target molecules4.3.1 TRIP plasmids,target plasmids and ubiquitin plasmids with different tags were co-transfected into HEK293T cells to detect the impact of TRIP on the ubiquitination of target molecules using co-immunoprecipitation technology.4.3.2 Control siRNA and TRIP siRNA were transfected into M619 cells respectively to detect its effect on the ubiquitination of target molecules.4.3.3 TRIP C5A plasmids with the mutation of the E3 ligase active sites,target molecular plasmids and ubiquitin plasmids with different tags were co-transfected into HEK293T cells,respectively,and the ubiquitination level of target molecular was detected using co-immunoprecipitation technology.4.3.4 TRIP,target plasmid and WT,K48 or K63 mutants of ubiquitin with different tags were co-transfected into HEK293T cells,respectively,to determine the specific form of TRIP regulating target molecule ubiquitination.4.3.5 Knockdown of TRIP in M619 cells to further determine the specific form of TRIP regulating target molecule ubiquitination.4.4 To investigate whether the E3 ligase activity effect of TRIP impact its biological function.Blank vectors,TRIP WT,C5A and △C51 plasmids were transfected into M619 cells,24 h later,the abilities of choroid melanoma cell proliferation and metastasis were detected by MTT and Transwell assays.Results:1.TRIP inhibits the proliferation and metastasis of choroid melanoma cells MTT and Transwell results showed that TRIP overexpression significantly inhibited the proliferation and metastasis of M619 and OCM-1 cells.On the contrary,TRIP knockdown significantly enhanced the proliferation and metastasis levels of M619 and OCM-1 cells.2.TRIP suppressed the EMT process of choroid melanoma cells RT-PCR and Western blot results showed that TRIP overexpression enhanced the expression of epithelial marker E-cadherin in M619 and OCM-1 cells,and reduce the expression of interstitial cell markers N-cadherin and Vimentin.TRIP knockdown can reduce the expression of epithelial marker E-cadherin in M619 and OCM-1 cells,and enhance the expression of mesenchymal markers N-cadherin and Vimentin.3.TRIP targets Twist13.1 Western blot results revealed that TRIP could specifically downregulate the expression of Twistl,while had no effect on the expressions of other EMT activating transcription factors(Snail,Slug,ZEB1).3.2 Co-immunoprecipitation results showed that TRIP could bind directly with Twistl,but not with other EMT-activating transcription factors.4.TRIP mediates the K48-linked ubiquitinated degradation of Twist14.1 Western blot results showed that TRIP mediated the degradation of Twist1 in a dose-dependent manner and depending its E3 ligase activity.4.2 The results of co-immunoprecipitation showed that TRIP combined with Twist1 through its N-terminal RING domain.4.3 Exogenous and endogenous ubiquitination experiments showed that TRIP could specifically mediate the K48-linked ubiquitination of Twistl,but had little influence on K63.5.The effect of TRIP on the proliferation and metastasis of choroidal melanoma cells depends on the E3 ligase activityMutation and deletion the E3 ligase active site could both neutralize the effect of TRIP on the proliferation and metastasis of choroid melanoma cells.Conclusion:1.TRIP inhibits the proliferation and metastasis of choroid melanoma cells.2.TRIP suppress the EMT process of choroid melanoma cells.3.TRIP targets Twistl and mediates its K48-linked ubiquitination degradation.4.The effect of TRIP on the proliferation and metastasis of choroidal melanoma cells depends on its activity of E3 ligase.Innovation and significances:1.TRIP can inhibit the proliferation and metastasis of choroidal melanoma cells and suppress the EMT process;TRIP can inhibit the expression of Twist1,but has little influence on the expression of other EMT activating transcription factors.These results have not been reported.2.Our study for the first time confirmed the regulatory role of TRIP in the proliferation and migration of choroid melanoma and clarified the molecular mechanism.TRIP was proposed and proved for the first time to regulate the occurrence and development of tumors through its E3 ligase activity,providing a new way to search for immunoregulatory therapy for gene transfection prevention and treatment of related malignant tumors.