Expression And Biological Significance Of MAP4K4in Bladder Cancer

ObjectiveBladder urothelial carcinoma(UC) is the most common malignancy of the urinary tract,70%bladder cancer are non-muscle-invasive but50%-70%of them will recur after surgery and10%-20%of them will progress into muscle-invasive bladder cancer(MIBC),1/3patients will died of MIBC even after radical cystectomy. Bladder cancer is one kind of common malignancy with high recurrence rate and relatively high mortality rate. Identifying bladder cancer associated oncogene or suppressor gene is of great interest for scientist and urologist, and bladder cancer management at the gene level is of great social and economical value.MethodsFirst,5pairs of fresh-frozen bladder cancer and adjacent normal mucosa were obtained from radical cystectomy specimen, total RNA were extracted and transcripted reversely to cDNA and then hybridized with gene chip containing25100gene probes to screen the differentially expressed genes.Next, To verify the result of microarray analysis at the level of mRNA and protein and clarify the clinical relevance of MAP4K4,16pairs of samples selected from fresh MIBC and adjacent tissues were detected respectively for the expression of MAP4K4mRNA and protein through RT-PCR, qRT-PCR and Western-blot. We depicted the expression profile of MAP4K4protein in156cases of paraffin embedded MIBC through imuno-histochemistry test. The expression level of MAP4K4protein was correlated with different clinicopathological parameters and Cox regression model was constructed to test its association with prognosis of MIBC after radical cystectomy.Finally, A control group and4groups of MAP4K4targeted shRNA were cotransfected with human bladder cancer cell line T24, stably transfected cells were tested regarding efficiency of knock down of target gene through qRT-PCR and Western-blot. Cells transfected with control shRNA and with shRNA of highest knockdown level were compared regarding cell shape, proliferation, invasion and apoptosis through microscopic observation, MTT, transwell and flowcytometry.ResultsAmong multiple differentially expressed genes, MAP4K4gene was concomitantly over-expressed among all5pairs of specimen.MAP4K4were overexpressed in bladder cancer than in adjacent tissue at both mRNA and protein level. The overexpression of MAP4K4protein in MIBC was correlated with lymphovascular invasion and positive lymphnode, weakly correlated with multicentricity and pathologic stage. Among lymphnode negative cases, MAP4K4overexpression was associated with poor prognosis after radical cystectomy.After target gene interference, the transfected cells become flattened, showed increased adherence to plate and growth retardation. Compared with control, cells transfected with the MAP4K4targeted shRNA Clone_TRCN32showed decreased proliferation rate, decreased invasiveness and increased apoptosisConelusionsMAP4K4is overexpressed in bladder cancer than adjacent tissue, and overexprssion of MAP4K4in MIBC is associated with poor prognosis after radical cystectomy, Knock down of MAP4K4in bladder cancer cell line T24retarded cell growth, inhibitted cell proliferation and invasiveness and accelerated cell apoptosis. MAP4K4is a target gene of gene therapy and worthy of further investigation.