Inhibition Of TGF-β By SiRNA Suppresses The Motility And Invasiveness Of T24 Bladder Cancer Cells Via Modulation Of Integrins And Matrix-metalloproteinase

Background:Bladder cancer is the fourth most common cancer in men and the five-year relative survival rates are about 6%if people are diagnosed with bladder cancer at a distant stage in American.Although chemotherapy has revolutionized the treatment of advanced tumors,the associated side effects induced by lack of specificity to tumor cells remain a challenging problem.TGF-β1 is a multifunctional cytokine which inhibits cell growth and also mediates cell differentiation and metastasis.Many studies have indicated that it is associated with epithelial-mesenchymal transition,angiogenesis,migration and metastases in many types of malignant tumors.Consistent with the idea that TGF-β1 can act as a oncologic promoter,increased expression of TGF-β1 in tumor cells and/or plasma has been correlated with advanced tumor progression in gastric carcinoma,human pancreatic carcinoma,hepatocellular carcinoma,adenocarcinoma and urinary bladder transitional-cell carcinoma.TGF-βhas been shown to induce the expression of matrix metalloproteinases(MMPs) which can degrade extracellular matrix allowing urinary bladder transitional-cell carcinoma cells to spread and diffusely infiltrate the bladder parenchyma.MMP-2 and MMP-9 in particular are consistently detected in malignant tissues and associated with tumor growth and progression.Integrins are a family of cellular adhesion molecules comprised two kinds of transmembrane subunits:αandβ, which are involved in a number of the cell-to-extracellular matrix(ECM) proteins and the cell-to-cell interactions.In mammals,18αand 8βsubunits associate in various combinations to form 24 integrins that can bind to various ECM ligands and it is well known that these integrins play pivotal roles in cell migration and adhesion in cancer cells.Recent studies showed that members of the integrinβ1 subfamily played an important role in tissue attachment,migration,invasion and metastasis of human cancers,and there was a correlation between increasedβ1-integrin expression and the more invasive and metastatic bladder cancer.Moreover,the expressions ofα5,αv integrin subunits andα3β1 integrin have been found being enhanced by TGF-β1 in cancers.Therefore,TGF-βsignaling pathway is emerging as an attractive target in cancer and inhibitors of this pathway may affect tumor progression and improve overall survival.TGF-βsignal propagates via specific TGF-βtypeⅠreceptor(TGFBRⅠ),which is activated by typeⅡreceptor(TGFBRⅡ).Activated TGFBRI initiates cytoplasmic signaling pathways via phosphorylation of Smad proteins,which are the main downstream components of TGF-βsignaling.Phosphorylated Smad proteins bind with the common Smad4 and translocate to the nucleus,where they interact with activators or repressors to mediate transcriptional regulation of target genes.Some approaches targeting TGF-βpathway have been developed,including blockade of TGF-βaction with neutralizing monoclonal antibody or RNAi,inhibition of TGF-βreceptor kinases or intracellular mediators by specific inhibitors.Anti-TGF-βstrategies inhibit the viability,migration and metastases of tumor cells in vitro.Objectives:The present study was undertaken to evaluate the effects of TGF-β1 and TsiRNA targets the TGF-βtypeⅠreceptor on the motility and invasiveness of T24 cell.We also examine the effects of TGF-β1 and TsiRNA on the expression of TGFBRI and tumor invasion and migration related genes in order to explore the possible mechanisms.Methods:In this study we blocked the TGF-βsignal pathway in T24 human bladder cancer cells with a siRNA(TsiRNA) which targets the TGF-βtypeⅠreceptor and evaluated the effects of TGF-β1 and TsiRNA on the cell motility and invasiveness by Matrigel migration assay,wound-healing assay and Matrigel invasion assay.RT-PCR and Western-blot analysis were used to examine the effects of TGF-β1 and TsiRNA on the expression of TGFBRI and tumor invasion related genes.All the detection items in this study were repeated at least 3 times.Statistical analysis was done using SPSS software.The data was expressed as mean±SD and the statistical significance of the differences between control and parthenolide-treated cells was determined by a two-tailed Student's t test.The Spearman correlation test was used to analyze the correlation between reagents' concentrations and inhibition rates.P-value＜0.05 was considered as significant.Results:Many reports have demonstrated that TGF-β1 promotes the motility of tumor cells in many types of tumors.Our study revealed an addition of TGF-β1(2 ng/ml) significantly enhanced the migration of the T24,while this enhanced migration could be effectively inhibited by TsiRNA.Similarly,exogenous TGF-β1(2 ng/ml) significantly enhanced the invasiveness of the T24 while TsiRNA evidently attenuated TGF-β1 induced invasion.Our results revealed that TGF-β1 enhanced the expression ofα3,β1 andα2 integrin subunits and MMP9,whereas TsiRNA suppressed these integrin subunits and MMP9,which may explain the diversion of motility and invasiveness of bladder cancer cell T24.Conclusions:In this study,all the results suggested that TGF-β1 was a major enhancer for the motility and invasiveness of bladder cancer cells via modulation of integrins and matrix-metalloproteinases.The TGFBRI targeted siRNA strongly inhibited the motility and invasiveness of the bladder cancer cells via down-regulation of the expression of integrins and matrixmetalloproteinase.Therefore,blockade of the TGF-βsignaling pathway by siRNA mediated knockdown of TGFBRI could be beneficial in the treatment of invasive bladder cancer.