The Preliminary Study Of The Functional Role Of NCAPH In The Carcinogenesis And Progression Of Bladder Cancer And Its Underlying Molecular Mechanism

Objectives:Bladder cancer is the most common malignant tumor in urinary system and the most common cause of cancer-related death around the world.Until recently,the treatment of bladder cancer,for several years,was limited to surgery and to immunotherapy or chemotherapy in clinical setting.Despite advanced progress in the early diagnose and treatment of bladder cancer has been made,approximately 50%patients occur recurrence and metastasis following surgery.Hence,exploring the pathogenesis of bladder cancer is important for the diagnosis and treatment in clinic.Non-SMC condensin I complex subunit H(NCAPH),is a multi-protein complex that plays a pivotal role in chromosome-wide gene regulation by controlling chromosome assembly and separation in the mitotic and meiotic cell cycles of proliferative cell.Numerous studies reported that NCAPH play an oncogenic role in the carcinogenesis of various solid tumors.For example,NCAPH affect the progression of colon cancer through promoting cell growth,migration,cell cycle progression and inhibiting apoptotic cell death.Similarly,NCAPH also enhances proliferation,migration,and invasion of hepatocellular carcinoma.However,no published literature has studied the functional role of NCAPH in the carcinogenesis and progression of bladder cancer.The data in TCGA database revealed that NCAPH was aberrantly up-regulated in bladder cancer tissues than that in matched-noncancerous tissues.Consequently,we speculated NCAPH might play a critical role in bladder cancer and attempted to explore its underlying mechanisms.Methods:1)30 cases fresh bladder urothelial cell carcinoma tissues and matched adjacent non-cancerous tissues,without preoperative radiotherapy or chemotherapy before operation,were collected,and total RNA were extracted from tissues and reversed-transcribed into c DNA.IHC and q RT-PCR were used to detected the m RNA expression of NCAPH in tumor tissues and normal tissues.The clinical information of those patients was collected and correlation between NCAPH level and clinicopathological features of patients was analyzed.2)Cell culture:Normal bladder epithelial cells(SV-HUC-1)and bladder cancer cell lines(UMUC-3,5637,SW780,and J82)were purchased from procell and icell biology life technology.SV-HUC-1 cells and 5637 cells were cultured in Ham's F-12K medium and RPMI-1640 supplemented with 10%FBS,respectively.SW780 cells were cultured with Leibovitz's L-15 medium contained 10%FBS.While UMUC-3 and J82 cells were cultured using MEM medium.All cells were incubated in a humidified incubator containing 5%CO₂at 37°C.3)The relative m RNA expression levels of NCAPH in bladder cancer tissues,normal bladder epithelial cells SV-HUC-1,and four bladder cancer cell lines(UMUC-3,5637,SW780,and J82)were analyzed using q RT-PCR.5.IHC was performed to examine the expression levels of NCAPH in bladder cancer tissues and adjacent non-cancerous tissues.4)Cell transfection:According to the q RT-PCR results,NCAPH overexpression plasmid and NCAPH si RNA(si NCAPH-1,si NCAPH-2,si NCAPH-3)were transfected into SW780 and UMUC-3 cells for 48h,respectively.Then MTT and Brd U assay were employed to examine the effect of NCAPH on cell proliferation of SW780 and UMUC-3.5)SW780 and UMUC-3 cells were routinely cultured and transfected with NCAPH overexpression plasmid and NCAPH si RNA(si NCAPH-1,si NCAPH-2)for 48h,respectively.Flow cytometry and TUNEL assay were used to examine the effect of NCAPH on cell apoptosis and cell cycle progression.While western blotting analysis was performed to determine the protein expression levels of cyclin B1,CDK1,MEK1/2,phospho-MEK1/2(ser217/221),ERK and phospho-ERF1/2(thr202/tyr204)6)NCAPH sh RNA stable transfection:Cell medium were replaced by fresh medium contained 500g/ml G418 to screen stable transfection cells,after transfection for 48h.Then q RT-PCR and western blotting were used to confirm the transfection of NCAPH-sh RNA in UMUC-3 cells.7)The mice were injected with NCAPH stably transfected UMUC-3 cells(1×10⁶cells)in their left flanks to establish bladder cancer xenograft model.Then the body weight and tumor volume of mice were measured twice every week until day 25.Results:1)q RT-PCR and IHC results suggest that NCAPH expression was significantly up-regulated in bladder cancer tissues than in normal bladder tissues(P<0.01).NCAPH expression could be detected in four bladder cancer cell lines(UMUC-3,5637,SW780,and J82),and NCAPH was obviously increased in bladder cancer cell lines,when compared with normal bladder epithelial cells(SV-HUC-1)(P<0.0001).Furthermore,in four bladder cancer cell lines,the relative expression level of NCAPH was highest in UMUC-3 cells,while SW780 was lowest.Hence,UMUC-3 and SW780 cells were selected as the research object in the following experiments.2)Correlation analysis results demonstrated that no obvious correlation between NCAPH level and sex,age of bladder cancer patients was found(P>0.05),while NCAPH level were positively correlated with tumor size(P=0.00382),stage(P=0.00382),and lymph node metastasis(P=0.03142).3)q RT-PCR and western blotting results revealed that the expression of NCAPH in NCAPH overexpression plasmid transfected SW780 cells was elevated 5-fold than that in control group(P<0.0001),while was significantly reduced in si NCAPH-1,si NCAPH-2,and si NCAPH-3 infected UMUC-3 cells.4)MTT and Brd U staining results indicated NCAPH overexpression could effectively promote cell growth of SW780 cells,while knockdown NCAPH with si RNA significantly reduce proliferative ability of UMUC-3 cells.5)Flow cytometry analysis and TUNEL staining showed that NCAPH overexpression significantly promoted cell cycle progression,and avoided apoptotic cell death in SW780 cells,while knockdown of NCAPH significantly induced cell cycle arrested at G2/M phase,and enhanced apoptotic cell death.6)Western blotting results showed that overexpression of NCAPH in SW780 cells effectively increased the protein expression level of cyclin B1,CDK1,phospho-MEK1/2(Ser217/221),and phospho-ERK1/2(Thr202/Tyr204),while knockdown NCAPH with si RNA in UMUC-3 cells showed the opposite effect.7)In vivo studies indicated that knockdown NCAPH using sh RNA could dramatically reduce the growth of tumor in bladder xenograft model and down-regulate the expression of ki-67 in tumor tissue.Conclusion:Our findings suggest that NCAPH might functions as a novel oncogene in the carcinogenesis and development of Bladder Urothelial Cell Carcinoma.NCAPH was aberrantly up-regulated in bladder cancer and were positively correlated with tumor size,stage,and lymph node metastasis.NCAPH overexpression could significantly promote cell proliferation and cell cycle progression,and avoid apoptotic cell death.Moreover,the activation of MEK/ERK signaling pathway was associated with the oncogenic role of NCAPH in bladder cancer.Collectively,NCAPH might represent a potential therapeutic target for the treatment of bladder cancer.