The Expression Of MTA1 Gene In Bladder Cancer And Its Effect On Biological Behavior Of Bladder Cancer Cell

Bladder transitional cell carcinoma is most common tumor in urothelial carcinoma,Although there are many new techniques coming out,,but tumor invasion and metastasis is the key cause of failure to cure and high mortality in bladde cancer.So,it may be necessary to understand the mocular mechamisms of invasion and metastasis of bladder transitional cell cacinoma.Here,we show the metastasis-associated gene 1(MTA1)could be another candidate factor that is associated with invasion and metastasis.Several research had found that a correlation between MTA1 gene overexpression and tumor invasiveness,but no studies on MTA1 had been performed on the bladder cancer,and no systematic biological studies had been performed on bladder cancer.Experimental methods and results were as follows.Part 1 Expression of MTA1 and the potential clinical implications in human bladder transitional cell carcinomaOBJECTIVE:The purpose of this study was to examine the mRNA and the protein levels of the MTA1 in human bladder transitional cell carcinoma and thus to evaluate the association of this gene with invasion and metastasis in bladder cancer.METHODS:The expression of MTA1 mRNA and protein in 34 bladder cancer samples was compared with that in corresponding normal mucosa tissues by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)and immunohistochemistry,the results were compared with clinicopathologic data. RESULTS:The expression of MTA1 mRNA was detected in all of 34 bladder cancer and corresponding normal mucosa tissues.A relative overexpressing of MTA1mRNA(tumor/normal ratio≥2)was observed in 14 of 34(41.2%)bladder cancer.Clinicopathologic correlations demonstrated that in human bladder transitional cell carcinoma overexpressing.MTA1-mRNA exhibited a significantly deeper wall invasion and a high rate of metastasis to lymph nodes.We found the MTA1 protein locates in the nucleus by immunohistochemistry and there was significantly associated with the expression of the MTA1 protein and tumor invision and lymph metastasis,but not with pathological grade.CONCLUSION:That overexpression of MTA1 was significantly correlated with tumor invasion and metastasis in bladder cancer strongly suggests the possibility that MTA1 is one of the key molecules associated with tumor invasion and metastasis.Part 2 Construction of MTA1-specific siRNA expression vectorsOBJECTIVE:To design a series of 19nt siRNA sequence targeting MTA1 and construct MTA1-specific siRNA expression vectors.METHODS:According to the computer aided design,the sequences of the siRNA for the MTA1 gene(MTA1-siRNA)were selected from nucleotide sequences 799-818(i-1,sense sequence:5′-GAACATCTACGACATCTCC-3′; antisense sequence:5′-GGAGATGTCGTAGATGTTC-3′), 1102-1120(i-2,sense sequence:5′-GGATTTCACGGACATTCAG-3′; antisense sequence:5′-CTGAATGTCCGTGAAATCC-3′),2020-2038(i-3 sensese quence:5′-GATCCGCAAGCTGCTCTCA-3′;antisense sequence: 5′-TGAGAGCAGCTTGCGGATC-3′)of MTA1 cDNA(GenBank accession numbers GI:115527079).A sequence non-specific to any known gene was used as a negative control,oligonucleotide fragments containing different MTA1 specific sequences were synthsized and cloned into the expression vector psiRNA,and four recombinant vectors shRNA-1,shRNA -2,shRNA-3 and shNeg were constructed. RESULTS:MTA1-specific siRNA expression vectors were confirmed by PCR analysis and the results of DNA sequencing was correct.CONCLUSION:The plasmid expressing shRNA homologous to MTA1 mRNA were constructed successfully using shRNA expressing vector pRNAT-U6.1.Part 3 The effect of shMAT1 on MTA1 gene expression in BIU-87OBJECTIVE:To investigate the effect of shMTA1 on MTA1 gene expression in BIU-87,and establish stable shMTA1-transfected BIU-87 cells.METHODS:Cultured BIU-87 cell were transfceted with MTA1 shRNA,and the three shRNA expression vetors were separately transfected into BIU-87 cells by Lipofectamine 2000,the cells treated with blank vetor served as controls.At the time of 48h posttransfection,total RNA was isolated and semi-quantified the MTA1mRNA expression levels were analyzed by RT-PCR.The optimal shMTA1 was selected for stable transfectant.The stable transfectant of BIU-87 cell acquired using G418 agent,the blocked expression of MTA1 in the stable transfectant was detected by RT-PCR and Western blotting.RESULTS:Using RT-PCR,we exhibitec that both shMTA1-1 and shMTA1-2 can knockdown MTA1 expression levels in specific manner,but shMTA1-3 was inefficient,vector shMTA1-2 can knockdown MTA1 expression more effectively compared with vector shMTA1-1.Subsequently, the shMTA1-2 and pRNAT-U6.1 expression vectors was separately transfected into BIU-87 cells by liposome method.After prolonged G418 selection,the transfected cells with shMTA1-2 and pRNAT-U6.1 were subject to a large scale cultivation.RT-PCR and Western blotting analysis showed the expression levels of MTA1 was significantly down-regulated in BIU-87 cells.CONCLUSION:The recombinant plasmid constructed can suppress the expression of MTA1 mRNA and protein in transfected cells,we had successfully established stable shMTA1-transfected BIU-87 cells.Part 4 The influences of MTA1 knockdown on cell biological behavior of BIU-7OBJECTIVE:To illustrate the possible influences of MTA1 knockdown on biological behavior and futher offer new theory reference and idea for the prevention and therapy of bladder cancer.METHODS:The experiment group was BIU-87 cells transfected with shMTA1-2,the controls were BIU-87 cells transfected with pRNAT-U6.1 vector and parental cell BIU-87.The effect of BIU-87 cell proliferation was estimated by cell growth curve,MTT and colony form assay,the cell cycle was analyzed by flow cytometric,(FCM).Millicell PCF insert and in vitro wound healing assay were performed to investigate the influence of MTA1 knockdown on BIU-87 cell invasion and mobility,cell-martrix adhenion was utilized to evaluate the effects of MTA1 gene on adhesion ability of cnacer cells.RESULTS:Compared with untransfected BIU-87 cells and pRNAT-U6.1 vector transfected BIU-87 cells,BIU-87-shMTA1-2 cells proliferated much more slowly indicating MTA1 silencing obviously affect cell proliferation.The ability to form colonies also was inhibitor compared to that parental BIU-87 and BIU-87 cells transfected with pRNAT-U6.1 vector.These results provided as evidences that the inhibitory effect on cell proliferation of knockdown MTA1 in BIU-87 cells.Flow cytometry(FCM) Analysis was performed,the results indicated that knockdown of MTA1 Could delay G1-S progression,cause cells arrest at G1 Phase,leading to an increase in the number of cells at G1 phase(77.47%vs50.18%/50.13%)and decrease in the number of cells at S phase(17.95%vs44.45%/41.82%) and G2/M phase(4.58%vs5.42%/8%).In the cellular biological studies,we show that MTA1 expression is positively correlated with in vitro mobility and invasion ability in bladder cancer cell line BIU-87.The ability of adhesion of BIU-87 cells transfected shMTA1-2 to extracellular matrix decrease compared with the parental BIU-87 and BIU-87 cells transfected with pRNAT-U6.1 vector,the results suggest MTA1 exert its effects on metastasis by regulating the adhesion of BIU-87 cells.CONCLUSION:Our results demonstrate that knockdown of MTA1 in BIU-87 cells by RNAi technique resulted in inhibition of cell proliferation and growth,reduction of cell mobility and invasion indicating MTA1 might be a potential bladder cancer metastasis associated candidate gene,and abnormal expression of MTA1 may play an important role in invasion and metastasis of BIU-87 cells.