The Mechanism Study Of Valproic Acid Inhibition Of Epithelial Mesenchymal Transition In Prostate Cancer Via The TIF1? Mediated TGF-?/Smad Signaling Pathway

Background:Prostate cancer(PCa)is a common malignant tumor of the urinary system.In the United States,PCa has become the first malignant tumor that endangers men's health,with the second highest mortality rate.The incidence of PCa in China is also increasing year by year.PCa has become one of the problems affecting the health of men in China.The progression of PCa is relatively slow.For localized and locally advanced PCa,the 5-year survival rate is greater than 99%,while the 5-year survival rate of advanced patients is significantly reduced to about 30%.The transformation from androgen-dependent prostate cancer(HSPC)to castration resistant prostate cancer(CRPC)and distant metastasis of the tumor are the main causes of poor treatment efficacy and reduced survival rate,and there is still a lack of effective treatments.Therefore,it is extremely important to inhibit the metastasis of prostate cancer and seek new therapeutic targets.Epithelial mesenchymal transition(EMT)refers to the phenomenon of epithelial cells transforming into mesenchymal cells under specific physiological or pathological conditions,which is usually characterized by up-regulation of mesenchymal cell markers such as vimentin,N-cadherin and down-regulation of epithelial cell markers such as E-cadherin.The occurrence of EMT in tumor cells can increase the ability of tumor cells to invade and metastasize,and eventually metastasize to bones,lungs and other parts,thereby promoting tumor progression.Studies have shown that EMT of prostate cancer plays a key role in tumor progression,metastasis and treatment resistance.Therefore,studying the role of EMT in PCa metastasis is of great significance for the treatment of PCa.EMT is usually involved in the regulation of multiple signaling pathways,such as TGF-?/Smad,Wnt and ERK signaling pathways.Among them,TGF-?/Smad signaling pathway is considered to be an important link in the occurrence of EMT.In the classic TGF-?/Smad signaling pathway,TGF-? binds to type I and type II receptors(T?RI and T?RII)with serine-threonine kinase activity,phosphorylates and activates Smad2 and Smad3(R-Smad),The Smad2/3 complex interacts with the Smad4(Co-Smad)protein to form a transcription complex,which is then transferred from the cytoplasm to the nucleus to regulate the transcription of target genes.Therefore,blocking the TGF-?/Smad signaling pathway can inhibit the EMT process of tumor cells,thus providing a target for the treatment of prostate cancer.Valproic acid(VPA)is a broad-spectrum antiepileptic drug currently used in the treatment of epilepsy in adults and children.VPA is a class I(Ia,?b)and class ?(?a)inhibitor of histone deacetylase(HD AC).Studies have shown that in addition to its powerful neurotransmitter regulation function,VPA can also play a role in tumor cell cycle regulation,cell differentiation,DNA repair,apoptosis and autophagy.Its potential anti-tumor effect has been confirmed by many studies.The results show that VPA can induce apoptosis in acute myeloid leukemia cells and has a strong antiproliferative effect on breast cancer cells.Mechanistically,VPA can inhibit the proliferation of gastric cancer cells by targeting HDAC1/2 and HDAC1/PTEN/AKT signaling pathway-mediated autophagy.Not only that,VPA can also downregulate the expression of EGFR,ErbB2 and ErbB3 by regulating various key miRNAs in pancreatic cancer cells,which leads to the inactivation of downstream AKT and MAPK signaling pathways,inducing apoptosis and growth arrest.The above studies reveal that VPA inhibits tumor progression through complex regulatory mechanisms.However,some researchers have found that VPA can promote the occurrence and development of tumors.Recent studies have shown that VPA can promote the EMT of breast cancer cells by stabilizing Snail and up-regulating the expression of ZEB1.Therefore,we speculate that the effect of VPA has obvious specificity,and the outcome may have a great correlation with tumor type,intervention method,specific action concentration,and treatment time.Our previous research confirmed that VPA can reduce the abundance of Smad4 protein expression and increase its mono-ubiquitination level,that is,VPA can inhibit the EMT of PCa cells through dual action.But how VPA participates in the regulation of Smad4 mono-ubiquitination,its mechanism of action is unclear.Transcription mediator 1?(TIF1?),also known as TRIM33,is a member of the E3 ubiquitin ligase family and is involved in DNA repair,cell cycle regulation,immune response,and inflammatory response.Related research reports that TIFly,as a signaling molecule in the TGF-? superfamily,plays an important role in theTGF-?/Smad signaling pathway.Related studies in breast cancer have shown that FOXM1 can prevent TIF1? monoubiquitinated Smad4 by competitively binding Smad3/4 with TIF1?,weakening the inhibitory effect of TIFly on the TGF-?signaling pathway,thereby promoting breast cancer metastasis.SOX2 can inhibit the protein expression of TIFly by inhibiting the activity of TIFly promoter in lung cancer cells,and promote the invasion of EMT induced by TGF-?/Smad signaling pathway in lung cancer.However,the study of TIFly in prostate cancer has not been reported.By retrieving relevant literature and combining our previous research foundations,we hypothesized that in prostate cancer cells,does TIFly also participate in the EMT,invasion and metastasis of PCa induced by TGF-?/Smad signaling pathway in a similar mechanism?And does VPA play a role in increasing Smad4 mono-ubiquitination through TIFI y?This deserves our further study.Therefore,this experiment is to explore whether TIFly is a key molecule for VPA to inhibit EMT of prostate cancer,and to further explore the role of TIFly on TGF-?/Smad signaling pathway and its molecular mechanism,and to further verify in vivo experiments,providing a new target for clinical treatment of prostate cancer.Objective:1.On the basis of further verification that VPA inhibits EMT of prostate cancer cells,the paper explored whether TIFly mediates this process,and in vivo experiments are conducted to further verify,thereby providing new ideas for the treatment of prostate cancer..2.To investigate the role of TIFly on Smad4 and its molecular mechanism,and to clarify its role in the TGF-?/Smad signaling pathway,so as to provide scientific basis for the clinical application of VPA in prostate cancer.Methods:Part 1:The mechanism study of valproic acid inhibition of epithelial mesenchymal transition in prostate cancer via the TIFly mediated TGF-?/Smad signaling pathway1.Study on the role of TIFly in the inhibition of prostate cancer EMT,invasion and migration by VPA1.1.Study on the effect of VPA on prostate cancer cell EMT Human prostate cancer cell lines were selected as the main research objects,including androgen-sensitive prostate cancer cells-LNCaP cells and androgen-resistant prostate cancer cells-PC3 cells.LNCaP and PC3 cells were cultured in vitro,and according to the previous research results of the research group,VPA intervention of 2.4 mmol/L was selected as the experimental condition for 48h.After the cell intervention,the protein expression of EMT markers E-cadherin,N-cadherin and vimentin was detected by Western Blot(WB)method,and the effect and influence of VPA on EMT of prostate cancer cells were analyzed.1.2 Effect of TIFly on EMT of prostate cancer cellsThe effect of TIFly on EMT of prostate cancer cells was explored by gene-regulation of TIFly.First,we construct the lentivirus with TIFly knockdown and overexpression.After lentivirus infects LNCaP and PC3 cells,puromycin and blasticidin were used for cell screening to establish a stable infected cell line.The infection efficiency was observed by high content microscope.qRT-PCR and WB experiments were used to detect the mRNA and protein expression of TIF1?.Finally,LNCaP and PC3 cells with the best effect were selected for stable experiments and subsequent experiments.At the same time,the negative control(NC)group was used as a virus infection control group.On this basis,in order to clarify the effect of TIFly in prostate cancer cell of EMT,the following groups were set:TIFly knockdown-related experiments were divided into control group(sh-NC)and TIFly knockdown group(sh-TIFly).TIF1?overexpression related experiments were divided into control group(NC)and TIF1?overexpression group(TIFly).In order to clarify the effect of TIF1? expression changes on EMT of PC3 and LNCaP cells,the protein expressions of E-cadherin,N-cadherin and vimentin in PC3 and LNCaP cells were detected by WB experiment.1.3.Effect of VPA on TIFly expressionLNCaP and PC3 cells were cultured in vitro,and different concentrations of VPA were used to investigate the effect of VPA on TIFly expression.The following experimental groupings were performed:control group,1.2 mmol/L group,and 2.4mmol/L group.After the cells were passaged,each experimental group was added with VPA for 48h according to the pre-designed concentration.The expression of TIFly protein was detected by WB method.The effect of VPA on the expression of TIFly protein was observed,and the appropriate concentration of VPA was selected as the observation point for subsequent studies according to the experimental results,1.4.The role of TIF1? in inhibiting EMT and migration,invasion of prostate cancer cells by VPAIn order to clarify the role of TIF1? in the inhibition of EMT of prostate cancer cells by VPA,we designed a rescue experiment for TIF1? based on VPA intervention.First,the TIF1? knockdown related experiments of LNCaP and PC3 cells were designed,and the following experimental groupings were conducted:control group(sh-NC),VPA intervention group(VPA),TIF1? knockdown group(sh-TIF1?),and TIF1? knockdown VPA intervention group(sh-TIF1?+VPA).After that,the experiments related to TIF1? overexpression of LNCaP and PC3 cells were designed,and the following experimental groupings were conducted:control group(NC),VPA intervention group(VPA),TIF1? overexpression group(TIF1?),TIF1?overexpression VPA intervention group(TIF1?+VPA).The cells of each group were cultured in vitro according to the requirements of the previous experiment,and the intervention group was given VPA intervention for 48h.In order to clarify the role of TIF1? in VPA inhibiting EMT of prostate cancer cells,the expression of E-cadherin,N-cadherin and vimentin in each group of EMT was detected by WB.Similarly,based on the VPA intervention,the transwell test was applied to detect the effect of TIF1? expression changes on VPA inhibiting prostate cancer cell migration and invasion.The experiment grouping is the same as above.The cells in each group were cultured in vitro.After 48 hours of intervention in the intervention group,the changes of cell migration and invasion in each group were observed by the the transwell experiment,thus clarifying the role of TIF1? in the inhibition of prostate cancer cell migration and invasion by VPA.2.Study of mechanism on TIF1?-mediated VPA inhibition of prostate cancer EMTMany studies have shown that TIF1? can participate in the TGF-?/Smad signaling pathway by regulating the level of Smad4 mono-ubiquitination and play a role in suppressing EMT of tumor cells.To further explore the mechanism of TIF1?-mediated VPA inhibiting EMT of prostate cancer,we conducted the following experiments to investigate whether TIF1? can promote the dissociation of Smad3/Smad4 by increasing the level of mono-ubiquitination of Smad4.The experimental groups are as follows:sh-NC group,VPA group,sh-TIF1? group,sh-TIF1?+VPA group.PC3 cells of each experimental group were cultured in vitro and given VPA intervention for 48h.Co-immunoprecipitation was first used to detect the effect of VPA intervention on the level of Smad4 mono-ubiquitination and the expression level of Smad3/Smad4 complex,and to determine whether VPA could promote the solution of Smad3/Smad4 complex by up-regulating Smad4 mono-ubiquitination.Furthermore,on the basis of VPA intervention,the expression of TIF1? was knocked down,and the co-immunoprecipitation method was used to detect the mono-ubiquitination level of Smad4 and the expression level of Smad3/Smad4 complex,so as to determine whether TIF1? is the key molecule of VPA inhibiting prostate cancer EMT and its molecular mechanism.That is whether VPA exerts its role of increasing the level of mono-ubiquitination of Smad4 and reducing the level of Smad3/Smad4 complex through TIF1?,thereby inhibiting the EMT of prostate cancer induced by TGF-?/Smad signaling.Part 2:Study of VPA inhibiting the proliferation of prostate cancer xenografts in nude mice by TIF1?1.Study of TIF1? mediating VPA on inhibiting the proliferation of prostate cancer xenograft tumor in nude mice.48 male nude mices of 3-4 weeks old were selected and randomly divided into 8 groups,each with 6 mices.The following experimental design was carried out:?TIF1? knockdown expression related experiments were divided into:sh-NC group,VPA group,sh-TIF1? group,sh-TIF1?+VPA group.?TIF1? overexpression related experiments were divided into NC group,VPA group,TIF1? group,TIF1?+VPA group.Prostate cancer PC3 cells,stably transfected PC3 cells with knockdown and overexpressing of TIF1? were transplanted in nude mice.The intervention group was given intragastric treatment with 0.4%(w/v)VPA,100ul each time,and the control group was given the same amount of sterile deionized water,the intervention time was 2 weeks.During tumor formation and VPA intervention,the long and short diameters of the tumor were measured twice a week,and the tumor volume was calculated.Finally,we observed the effect of VPA intervention on the proliferation of transplanted tumors and whether the change of TIF1? expression affects the effect of VPA on the transplanted tumors.2.Study of TIF1? mediating VPA on inhibiting the EMT of prostate cancer xenograft tumor in nude mice.2.1.Effect of VPA on the EMT of prostate cancer xenografts in nude miceUsing the above transplanted tumor tissue samples,the WB experiment was used to detect the expression changes of E-cadherin,N-cadherin and vimentin between NC group and VPA group or between sh-NC group and VPA group.The semi-quantitative analysis and comparison between each group were conducted to explore the effect of VPA on the transplanted tumor EMT of nude mice..2.2.Effect of TIF1? on EMT of prostate cancer xenografts in nude miceThe WB experiment was applied to detect the expression changes of E-cadherin,N-cadherin and vimentin in the sh-NC group,sh-TIF1? group,NC group,and TIF1?group.The semi-quantitative analysis and comparison between each group were conducted to explore the changes in TIF1? expression on effects of EMT.2.3.Effect of VPA on TIF1? expression of prostate cancer xenograftsIn order to explore the effect of VPA on the expression of TIF1? in nude mice,WB experiment was used to detect the abundance of TIF1? expression in NC group,VPA group or sh-NC group,VPA group,and semi-quantitative analysis was conducted to determine the expression of TIF1? in transplanted tumor tissue by VPA intervention.2.4.Effect of TIF1? on VPA inhibiting EMT of prostate cancer xenograftsIn order to clarify the effect of changes in the expression of TIF1? on VPA inhibiting EMT of prostate cancer in the transplanted tumor tissue,the experimental groups were designed as follows:?TIF1? knockdown expression related experiment groups were:sh-NC group,VPA group,sh-TIF1? group,sh-TIF1?+VPA group;?TIF1? overexpression related experiments were divided into NC group,VPA group,TIF1? group,TIF1?+VPA group.The WB experiment was applied to detect the EMT-related indicators of each experimental group,including the expression of E-cadherin,N-cadherin,vimentin,and to observe the effect of TIFly knockdown or overexpression on the effect of VPA on inhibiting EMT in nude mice.At the same time,immunohistochemistry(IHC)was used to detect the transplanted tumor tissue samples of nude mice.The expression of TIF1?,E-cadherin,N-cadherin,vimentin in each experimental group was observed.The effects of VPA and TIF1?on the EMT of transplanted tumors,the effects of VPA on TIF1? were detected.The expression changes of TIF1? on the effect of VPA with the inhibition of transplanted tumor EMT were also detected.Results:Part 1:VPA inhibited the EMT in prostate cancer via TIF1?-mediated TGF-?/Smad signaling pathway1-TIFly mediated the process of VPA inhibiting prostate cancer cell EMT1.1.VPA could inhibit EMT of prostate cancer cellsAfter 48 hours of VPA intervention in LNCaP and PC3 cells at a concentration of 2.4 mmol/L,the protein expression of E-cadherin,N-cadherin and vimentin was determined by WB experiment.The results showed that after VPA intervention,the expression of E-cadherin in LNCaP and PC3 cells was significantly up-regulated,and the expression of N-cadherin and vimentin was significantly down-regulated.Compared with the control group,the difference was statistically significant.The above results indicated that VPA could inhibit the EMT of prostate cancer cells and play an important anti-tumor effect.1.2.TIF1? played an important role in inhibiting prostate cancer cell EMTThe stable transfection of LNCaP and PC3 cells with knockdown and overexpression of TIF1? were successfully constructed,and the transfection efficiency was higher than 90%by high content microscopy.qRT-PCR and WB methods were used to detect the expression of TIF1? mRNA and protein.The results showed that the knockdown efficiency was greater than 80%and the overexpression efficiency was greater than 150%.Comparing the knockdown and overexpression efficiency of each stable infected cell line,sh-TIFly-2 and TIF1?-3 were selected for LNCaP cells,sh-TIF1?-2 and TIF1?-2 were selected for PC3 cells for subsequent experiments.WB experiment was used to detect the effect of knockdown or overexpression of TIF1? on EMT of LNCaP and PC3 cells.The results showed that knocking down the expression of TIF1? could down-regulate the expression of E-cadherin in LNCaP and PC3 cells,and up-regulate the expression of N-cadherin and vimentin.Compared with the sh-NC group,the difference was statistically significant.Overexpression of TIF1? could up-regulate the expression of E-cadherin in LNCaP and PC3 cells,and down-regulate the expression of N-cadherin and vimentin.Compared with NC group,the difference was statistically significant.This indicated that TIF1? played an inhibitory role in EMT of prostate cancer cells.1.3.VPA could up-regulate the protein expression of TIF1? in prostate cancer cellsAfter 48 hours of intervention with different concentrations of VPA in LNCaP and PC3 cells,WB experiment was used to detect the protein expression of TIF1?.The results showed that VPA could significantly increase the expression of TIF1? protein in PC3 and LNCaP cells of prostate cancer,and it was concentration dependent.When the concentration of VPA was 2.4 mmol/L,the effect was most obvious.1.4.TIF1? mediated the process of VPA inhibiting prostate cancer cell EMTTo clarify the effect of TIF1? on VPA inhibiting EMT of prostate cancer cells,the expression of TIF1? was knocked down,and the changes of E-cadherin,N-cadherin and vimentin expression were observed by WB experiment.The results showed that in LNCaP and PC3 cells,after knocking down the expression of TIF1?,the intervention of VPA did not up-regulate the expression of E-cadherin and down-regulate the expression of N-cadherin and vimentin,indicating that knocking down the expression of TIF1? weakened the inhibition of VPA on the EMT.On the contrary,after overexpression of TIF1?,the up-regulation of E-cadherin and the down-regulation of N-cadherin and vimentin were more obvious based on VPA intervention,indicating that overexpression of TIF1? enhanced the effect of VPA on inhibiting EMT.The above data showed that VPA might inhibit EMT of prostate cancer cells through TIF1?.Cell migration and invasion experiments also showed that VPA could inhibit the migration and invasion ability of LNCaP and PC3 cells.After knocking down the expression of TIF1?,the migration and invasion ability of LNCaP and PC3 cells were significantly enhanced.After overexpression of TIF1?,the migration and invasion ability were inhibited.After knockdown expression of TIF1?,the effect of VPA on inhibiting the migration and invasion of prostate cancer cells was weakened,while overexpression of TIF1?,the effect of VPA on enhancing the migration and invasion of prostate cancer cells was enhanced.These phenomena also indicated that VPA could inhibit the migration and invasion of prostate cancer cells through TIF1?.2.TIFly mediated the inhibition of VPA on the EMT of prostate cancer cells via TGF-?/Smad signaling pathwayTo investigate whether TIF1? could inhibit the TGF-?/Smad signaling pathway by increasing the level of mono-ubiquitination of Smad4 and promoting the dissociation of Smad3/Smad4 and mediate the role of VPA in inhibiting EMT of prostate cancer cells,the mono-ubiquitination of Smad4 and Smad3/Smad4 complex levels were detected by co-immunoprecipitation.The results showed that the intervention of VPA could increase the level of mono-ubiquitination of Smad4 in PC3 cells and decrease the level of Smad3/Smad4 complex.Knocking down the expression of TIF1? reduced the level of Smad4 mono-ubiquitination and increased the level of Smad3/Smad4 complex.On the basis of VPA intervention,when the expression of TIF1? was knocked down,there was no increase in the level of Smad4 mono-ubiquitination and decrease in the level of Smad3/Smad4 complex,indicating that the down-regulation of TIF1? expression blocked the effect of VPA.Therefore,we speculated that TIF1? could play an important role in the process of VPA inhibiting EMT of prostate cancer cells through the TGF-?/Smad signaling pathway.Part 2:VPA inhibited proliferation and EMT of prostate cancer xenografts in nude mice via TIF1?1.TIF1? mediated the inhibition of VPA on the proliferation of prostate cancer xenograftsThe long and short diameters of transplanted tumors of nude mice in each experimental group were measured,and the tumor volumes of tumor-bearing mice in each experimental group were compared.The results showed that the average tumor volume of the control group was 0.72±0.12cm3,and the average tumor volume after2 weeks of VPA intervention was 0.44±0.11cm3.The difference was statistically significant,indicating that VPA could inhibit the proliferation of transplanted tumors.The tumor volume of the sh-TIF1? group increased significantly,with an average of 0.91±0.24cm3.Compared with the sh-NC group,the difference was statistically significant.The tumor volume of the TIF1? group decreased significantly,with an average of 0.48±0.09cm3.Compared with the NC group,it has statistical significance,indicating that TIF1? could inhibit the proliferation of transplanted tumors in nude mice.The average volume of tumors in the sh-TIF1?+VPA group was 0.54 ± 0.10cm3.Compared with the VPA group,the difference was statistically significant,indicating that the down-regulated expression of TIF1? weakened the proliferation inhibition effect of VPA.While the average volume of tumors in TIF1?+VPA group was 0.37 ±0.04cm3.Compared with the VPA group and TIF1? group,the difference was statistically significant,indicating that the overexpression of TIF1? promoted the proliferation inhibition effect of VPA.The above results indicated that VPA could inhibit the proliferation of PC3 cell xenografts in nude mice,and TIF1? mediated the proliferation inhibition effect of VPA.2.TIF1? mediated the inhibition of VPA on the EMT in prostate cancer xenografts2.1.VPA inhibited the EMT of prostate cancer xenografts in nude miceUsing PC3 cell transplanted tumor specimens from nude mice,WB experiments were carried out to observe the expression changes of E-cadherin,N-cadherin and vimentin after VPA intervention.The results showed that after VPA intervention,the expression of E-cadherin was significantly increased,and the expression of N-cadherin and vimentin were significantly down-regulated.Compared with the control group,the difference was statistically significant.It shows that in vivo experiments,VPA could also play its role in inhibiting EMT of prostate cancer.2.2.TIF1? inhibited the EMT of prostate cancer xenografts in nude miceIn order to further observe the effect of TIF1? on EMT in transplanted tumors,the WB experiment was applied to detect the expression changes of E-cadherin,N-cadherin,and vimentin after knockdown and overexpression of TIF1?.The results showed that after TIF1? knockdown,the expression of E-cadherin was significantly reduced,and the expression of N-cadherin and vimentin was significantly increased,indicating that inhibiting the expression of TIF1? could promote the EMT of transplanted tumors.Conversely,overexpression of TIF1? up-regulated the expression of E-cadherin and down-regulated the expression of N-cadherin and vimentin,indicating that the overexpression of TIF1? inhibited the EMT of the transplanted tumor.The above results indicated that in vivo experiments,TIF1?could inhibit EMT of prostate cancer as a tumor suppressor.2.3.VPA could upregulate the expression of TIF1? in prostate cancer xenografts To further clarify the effect of VPA on the expression of TIF1? in vivo,we compared the protein of TIF1? in the transplanted tumor tissue of the control group and the VPA group.WB experiment results showed that VPA intervention could significantly increase the protein expression of TIF1?.The difference was statistically significant comparing with the control group.2.4.TIF1? mediated the role of VPA in inhibiting EMT of prostate cancer xenografts in nude miceWB experiment was used to detect the protein expressions of E-cadherin,N-cadherin and vimentin in each experimental group.To further clarify the effect of TIF1?expression changes on EMT inhibiting of VPA,we compared the impact of TIF1? on the above indicators based on VPA intervention.The results showed that in the sh-TIF1?+VPA group,after suppressing the expression of TIF1?,there was no significant up-regulation of E-cadherin expression and down-regulation of N-cadherin and vimentin expression after VPA intervention.Compared with the VPA group,the difference was statistically significant significance,indicating that the down-regulation of TIF1? expression may block the effect of VPA on inhibiting EMT.In the TIF1?+VPA groups when VPA intervened in transplanted tumors overexpressing TIF1?,the up-regulation of E-cadherin and the down-regulation of N-cadherin and vimentin were more obvious,indicating that the up-regulation of TIF1? expression promoted the VPA inhibition effect of EMT.Similarly,the immunohistochemistry experiment also got similar results with WB.The above results suggested that TIF1? mediated the process of VPA inhibiting PC3 cell xenografts EMT in nude mice.Conclusions:1.The study clarified the role of VPA in inhibiting EMT of prostate cancer cells and revealed that TIF1? was the key molecule for VPA to inhibit EMT of prostate cancer.The in vivo experiments further confirmed that TIF1? mediated the inhibition of VPA on the proliferation of prostate cancer xenografts and EMT in nude mice.2.TIF1? regulated the EMT of prostate cancer induced by TGF-?/Smad signaling pathway by promoting the mono-ubiquitination of Smad4 and then inhibiting the formation of Smad3/Smad4 complex in prostate cancer cell.