| SUMO(small ubiquitin-like modifier)plays important roles in regulating protein functions and localization.SUMO modification is a dynamically reversible covalent modification process that can be reversed by SUMO-specific cysteine proteases(SENPs).SENPs are the pivotal proteins to maintain physiological functions via regulating the dynamic balance of SUMO modification.The abnormal expression of SENPs can destroy the balance of SUMO modification in vivo,which resutes in tumor development and progression.It has been reported that SENP1 is up-regulated in prostate cancer patients.Moreover,SENP1 interfering inhibits the bone metastases of high metastatic prostate cancer cells.However,the mechanisms of SENP1 mediated bone metastases of prostate cancer are still unknown.Transforming growth factor β(TGFβ)has been known to be an effective inducer of epithelial mesenchymal transition(EMT).In cancer cells,EMT is a pivotal process to promote development and progression of tumor cells.SMAD4 a tumor suppressor molecule,is a key regulator of TGFβ/SMADs growth inhibition signal.Inactivation of SMAD4 is closely related to the development and progression of prostate cancer.SMAD4 contains two SUMO location,SUMO modification can improve intracellular SMAD4 protein expression and enhance its stability,by avoiding ubiquitination degradation.Further studies have shown that SUMO-modified SMAD4 not only enhances TGFβ/SMADs mediated transcriptional activity,but also negatively regulates transcription factors,such as androgen receptors.Therefore,SUMO modification of SMAD4 may be involved in the development,progression and metastasis of prostate cancer.In the study,we hypothesized that the high expression of SENP1 in prostate cancer cells may promote EMT of tumor cells by deSUMOylating SMAD4,which in turn promote the development,progression and mestastases of prostate cancer.In this study,we try to investigate the influence of SENP1 on EMT and biological characteristics of prostate cancer cells,and to explore the roles of SENP1 in the regulating TGFβ/SMADs signaling.This study will provide new therapeutic targets and strategies for treatment of prostate cancer.Firstly,we designed and constructed recombinant lentiviral vector,PLKO.1-sh SENP1 to interfer expression of SENP1 in prostate cancer cells with high metastatic characteristics.And,we also constructed recombinant adenovirus,Ad-SENP1,to over-express prostate cancer with low metastatic potential.⑴ Construction of lentiviral vectors: The shRNA sequence targeting SENP1(shSENP1)and control shRNA(shscramble)was designed and synthesized.The shSENP1 and shScramble were inserted into PLKO.1 plasmids,and then the recombinant lentiviral vectors,PLKO.1-shSENP1 and PLKO.1-shScramble,were prepared by three plamids co-transfecting system.The recombinant lentiviruses,PLKO.1-sh SENP1 and PLKO.1-sh Scramble were purified with PEG,and the infection titers were 6.8×108 transducing units per milliliter(TU/ml)and 6.4×108 TU/ml,respectively.⑵ Construction of adenoviral vectors: The sequence containing open reading frame of SENP1 inserted into pShuttle-cmv to generate pShuttle-cmv-SENP1.Recombinant adenoviral vectors,Ad-SENP1 and Ad-Null,were constructed by Ad-easy system.Adenoviruses were purified by density gradient centrifugation of cesium chloride(CsCl).The infection titers of Ad-SENP1 and Ad-Null were 2.5×1010 IU/ml and 2×1010 IU/ml,respectively.Secondly,hormone-independent prostate cancer cells with high metastatic characteristics,PC3 M and PC3,were infected with 20 MOI lentiviral vectors,PLKO.1-sh Scramble or PLKO.1-shSENP1.Then,the proliferation,apoptosis and migration ability were examined;the markers of EMT,such as E-cadherin and Vimentin were detected;the TGF-β /SMADs signaling pathway correlated proteins,such as SMAD2/3 and SMAD4 were analyzed;SMAD4 regulated genes,including P15Ink4 B,P21WAF/Cip1,VEGF and HIF1? were also investigated.⑴After infected with PLKO.1-shSENP1 or PLKO.1-shscramble,the results of flow cytometry showed that silencing of SENP1 promoted cellular apoptosis,and inhibit proliferation of PC3 M cells(p<0.01).And,wound healing assay suggested that SENP1 silencing inhibited migration of PC3 M cells.⑵ Western-blotting showed that mediated interference of SENP1 could suppress EMT via up-regulating E-cadherin expression and down-regulating Vimentin expression at protein level(p<0.01).These results suggested that SENP1 silencing could inhibit the EMT of prostate cancer cells.⑶ We also showed that interference of SENP1 could increase the SMAD2/3 and SMAD4 expression at protein level in PC3 M cells.However,results of real-time RT-PCR showed that interference of SENP1 could down-rgulate SMAD2/3 and SMAD4 expression(p<0.01).These results indicated that SENP1 silencing regulated the SMAD2/3 and SMAD4 expression at post-transciptional level.⑷ In PC3 M cells,SENP1 interfernce reduced the expression of tumor metastasis related genes,such as VEGFA and HIF-1?(p<0.01,vs PLKO.1-shscramble group).Moreover,SENP1 interference also could inhibit the EMT of PC3 cells.Thirdly,in hormone-dependent prostate cancer cells with low metastatic potential,LNCaP,the EMT related molecules,TGF-β/SMADs cell signal correlated molecules,and SMAD4 regualted molecules were detected by western-blotting and real-time PCR,after infection with 10 MOI adenoviruses Ad-SENP1 or Ad-null.⑴ The results of western-blotting showed that Ad-SENP1 infection produced high level of SENP1 in LNCaP cells,and down-regulated the protein expression of SMAD2/3 and SMAD4.However,SENP1 over-expresion improved the mRNA expression of SMAD2/3 and SMAD4(p<0.01).⑵ Over-expressing SENP1 in LNCaP cells decreased the E-cadherin expression,while up-regualted the Vimentin expression,suggested that SENP1 could promte the EMT of prostate cancer cells.⑶ Morevoer,over-expression of SENP1 also could up-regulate HIF-1? expression,and down-regulate expression of pro-apoptosis genes(p<0.01,vs Ad-null group).These results suggested that SENP1 over-expression could promote the EMT and metastasis of prostate cancer cells.Finally,we constructed a plamid expressing SMAD4,and another plasmid encoding mutSMAD4,in which two SUMOylation sites were mutated.Then,they were transfected into SENP1 over-expressed prostate cancer cells to analyze the expression and transcriptional activity of SMAD4,and to analyze its effects on EMT of tumor cells.Moreover,we silenced the SMAD4 expression in SENP1 interfered prostate cancer cells by using siRNA targeting SMAD4.Then,the expression and transcriptional activity,and EMT of EMT were analyzed.⑴ Construction of pcDNA3.0-SMAD4 and pcDNA3.0-mutSMAD4.We synthesized the coding sequence of SMAD4,and then inserted into pcDNA3.0 to generate pcDNA3.0-SMAD4.The SUMOylation sites mutated SMAD4(K51R,K159R)were obtained by using over-lapping PCR,and the mutated SMAD4 was inserted into pcDNA3.0 to generate pcDNA3.0-mutSMAD4.⑵ The effects of SENP1 on the deSUMOylation of SMAD4.LNCaP cells were transfected with pcDNA3.0-SMAD4 and pcDNA3.0-mutSMAD4.Twenty-four hours later,cells were infected with recombinant adenoviruses,Ad-SENP1 and Ad-null,the corresponding proteins were detected by western-blotting.Our results showed that SENP1 down-regulated protein expression of SMAD4.However,mutated SMAD4 could inhibit SENP1 mediated down-regulation of E-cadherin.These results suggested that SENP1 might regulate E-cadherin expression via SMAD4.⑶ We transfected SENP1 silenced prostate cancer cells,PC3 M,with siRNA targeting SMAD4,and then the protein expression of SMAD4,E-cadherin and Vimentin were analyzed by western-blotting.Our data showed that interfering SMAD4 could inhibit the effects of SENP1 silencing on E-cadherin and Vimentin(p<0.01 vs siRNA-NC group).Taken together,SENP1 could regulate the deSUMOylation of SMAD4,which in turn regulate the expression of E-cadherin and Vimentin,and promote the EMT of tumor cells and tumor metastasis.In conclusion,we successfully constructed lentiviral vector expressing short interference RNA targeting SENP1(shSENP1)and adenoviral vector expressing SENP1 protein by using lentivial and adenoviral systems established in our laboratory.In prostate cancer cells with high metastatic characteristics,we showed that SENP1 interfence could inhibit the proliferation and migration of tumor cells,promote the apoptosis of tumor cells,activate TGF-β/SMADs signals and inhibit the EMT of tumor cells.Moreover,SMAD4 silencing could abolish the effects of SENP1 on expression of EMT related molecules,suggested that SENP1 silencing inhibit the EMT of tumor cells via regulating SMAD4.In prostate cancer cells with low metastatic potential,over-expressing SENP1 could down-regulate the SMAD4 expression and promote the EMT of tumor cells.However,transfection with mutSMAD4,which have two mutated sites at SUMOylation sites,could inhibit SENP1 induced EMT of tumor cells.These results suggested that SENP1 promote the EMT of tumor cells via deSUMOylating SMAD4.Therefore,SENP1 is a potential target for treating advanced and metastatic prostate cancer.And,our study will provide experimental for developing novel therapeutic strategy. |
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