| Prostate cancer is one of the major causes of cancer-related death in the western world.To date,it was still unclear about how to cure the advanced prostate cancer.Most of the prostate cancer sufferers were dead of the cancer migration.Epithelial Mesenchymal transition(EMT)is the first step in most cancers.A large number of researches showed EMT plays a crucial role in cancer progression.In prostate cancer metastasis,dysfunction of transforming growth factor-?1(TGF-?1)signaling pathway plays a pivotal role.Elevated TGF-?1 levels are detected in prostate cancer,and it seems to be related to tumor invasion.TGF-?1/Smads pathway is known as the canonical pathway.The Smad signaling crosstalk with other intracellular pathways leading to prostate cancer incidence and progression has been found in previous studies.Losing of growth inhibition of TGF-?1/Smads pathway and Epithelial-Mesenchymal Transition(EMT)underlying cancer cell invasion and metastasis in prostate cancer are the focuses of recent studies.Smad3 is one of the important cell signaling factors,which cooperate with Smad2 then transform TGF-?1 signaling into cytoplasm to regulate the expression of downstream molecules.Thus,disorder of Smad3 expression and activation may have significant correlation with the tumor genesis and deterioration in tumor progress.Notably:Although,a large body of evidence demonstrates that TGF-?1/Smads signaling contribute to the acquisition of the EMT and cancer metastasis,it is still unclear about the role of Smad3 in prostate cancer.In addition,the relationship between Smad3 and AR is inconsistent in across passages.Hence,we hope to demonstrate the efficiencies of Smad3 in cell proliferation,adhesion,invasion and migration ability of Hormonal Refractory Prostate Cancer cell line-PC-3 through investigate the EMT induce role and interaction of AR and Smad3 in PC-3 cell line.In the present study,Adhesion assay?Wound healing assay and Transwell assay were emplayed to investigate the alteration of cell adhesion,migration and invasion ability.It shows that after pSmad3,all of them were enhanced,on the contrary of siSmad3 in PC-3 cell line.Further study found that after stimulated with 1 ng/mL TGF-?1,adhesion,migration and invasion ability were enhanced in PC-3-pSmad3,compared with PC-3-WT and PC-3-Vector.However,there is no significant phenotypic modulation in PC-3-siSmad3 after exposed in 1 ng/mL TGF-?1.Additionally,we confirmed Smad3 contributes to the expression levels of EMT markers,Cancer Stem Cells(CSCs)markers and adhesion related cytokines such as ICAM-1,Rhoc-1,Axl.Real time Q-PCR analysis showed a marked induction of gene expression such as SOX2,Notch-1,Snail1,Slug,SIP1,ZEB1,N-cadherin and Vimentin after Smad3 overexpression in PC-3 in contrast to the cells treated with siSmad3.Moreover,it has also been shown that Smad3 was related to cell growth and apoptosis.MTT assay was found cell growth inhibition in PC-3 after specifically knockout of Smad3.Co-transfection with CKD2?CDK6 or CyclinD1 with siSmad3 could not rescue the anti-proliferation role caused by low level protein expression of Smad3.Besides,flow cytometry(FCM)was used to analysis cell apoptosis.Overexpression Smad3 induced an anti-apoptosis activity in PC-3.After stimulated PC-3 with 1 ng/mL TGF-?1,cell apoptosis was discovered.Reversely,we also implicated apoptosis inhibition phenomenon in PC-3-siSmad3,whereas PC-3 knockout of Smad3,the apoptosis inducing effect of TGF-?1 was inhibited.Based on these results,we conclude that Smad3 is a critical signaling molecule in PC-3 cell line.On the one hand,it can mediate EMT incidence through change the adhesion,migration and invasion ability of PC-3 cell line in TGF-?1 dependent or independent Smad3 signaling pathway.On the other hand,we demonstrate that Smad3 mediates cell apoptosis in a TGF-?1 independent manner,as well as influences the level of Cyclins and CDKs to medicate cell proliferation.This data provide a potential therapeutic target for HRPC treatment. |
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