The Study On The Invasion-promoting Role Of Zonula Occludens-1 Associated Nucleic Acid Binding Protein (ZONAB) In Bladder Cancer

Objective: Tight junction is the primary barrier against invasion and metastasis of cancer cells.Cancer cells lose intercellular junctions,dissociate from the original tumor site,and then invade into the surrounding tissue.Zonula occludens-1 associated nucleic acid binding protein(ZONAB)is an important tight junction protein and is also a transcription factor.When cell density increases,ZONAB in an inactivated state binds to zonula occludens-1(ZO-1).When ZONAB is activated,it goes into nucleus,specifically binds to the promoter of cyclinD1 and proliferating cell nuclear antigen(PCNA),and plays a role in promoting cell proliferation.ZONAB expression in liver cancer and colon cancer is upregulated to promote cancer cell proliferation.The expression pattern of ZONAB in bladder cancer is not well studied yet.Although the role of ZONAB as a transcription factor to promote cell proliferation has been reported,but whether it can affect tumor invasion is still unclear.The purpose of this study is to detect the alteration of ZONAB expression in bladder cancer and to demonstrate the role of ZONAB expression on cancer invasion,so that to reveal the potential mechanisms of bladder cancer invasion by gene transduction,in vitro and in vivo experimental methods.Methods:1.Subjects of the study: Patients admitted to the department of Urology in Shengjing affiliated Hospital of China Medical University and underwent surgery between 2014 and 2016 were selected.30 cases of muscle-invasive bladder cancer,41 patients with non-muscle-invasive bladder cancer and corresponding adjacent non-tumor urothelial tissues were collected,whose pathological diagnosis was urothelial carcinoma.Urinary tract infection was excluded.Tumor tissue and adjacent non-tumor tissues from surgery were obtained,from which one portion was fixed and sent to pathological examination and immunohistochemical experiment and another portion was transferred and stored into a-80? refrigerator.Immortalized human urothelial cell line sv-huc-1 and urothelial carcinoma cell line 5637,T24,UM-UC-3 and were purchased from China Center for Type Culture Collection of Chinese Academy of Sciences(Shanghai).2.Construction of polyclonal T24 cell line with ZONAB overexpression: LB medium containing ampicillin was used to culture pLV-ZONAB-DU and control group pLV-GFP-DU bacteria.The bacteria were collected and plasmids were extracted.UV spectrophotometer was used to measure OD260 value and calculate the concentration of the plasmids.The lentiviral vector,pH1,pH2 were used to infect HEK293 T cells.Supernatants were collected and viruses were purified.Lentivirus suspension was added to complete medium with T24 cell growing,followed by adding polybrene to a final concentration.The original medium was replaced with a viral infection medium.Cells were frozen after 14 days.3.Transwell chamber invasion assay: Diluted Matrigel was added into the upper chamber of the Transwell culture dish to cover the polycarbonate membrane.Single cell suspension was added into the upper chamber of the Transwell culture dish.Chemokines were added into the lower chambers of the Transwell culture dish.After 1 day culture,cells on Matrigel gel and polycarbonate membrane surface were wiped off.The upper chamber was fixed with methanol,stained with Hematoxylin,dehydrated in a graded ethanol and cleared with xylene.Polycarbonate membrane was cut from the upper chamber,placed on the slides,and mounted with a neutral resin.The cells were counted under high magnification.4.Preparation of animal model: Each group of human bladder cancer cells was injected subcutaneously in both axillary flank portion of the nude mice.Each diameter of the tumor was measured.5.Westem blotting: It was used to detect the specific protein expression levels in bladder tissue and each cultured cell line.6.realtime-PCR: It was used to detect the specific mRNA expression levels in bladder tissue and each cultured cell line.7.Immunohistochemical staining: It was used for observation and detection of specific protein expression state and levels in bladder tissue.8.H & E staining: It was used for observation primary tumor invasion in bladder cancer animal models.Result:1.ZONAB expression is upregulated in bladder cancer(1)Detection of ZONAB mRNA expression in bladder cancer and adjacent non-cancerous tumor tissues by realtime-PCR: ZONAB mRNA expression in bladder cancer was higher than that in adjacent non-tumor tissue.(2)Detection of ZONAB protein expression in bladder cancer and adjacent non-tumor tissues by Western blotting: ZONAB protein expression in bladder cancer was higher than that in adjacent non-tumor tissue.(3)Detection of ZONAB protein expression in bladder cancer and adjacent non-cancerous tumor tissues by immunohistochemical staining: High staining rate of ZONAB protein in muscle-invasive bladder cancer,non-muscle-invasive bladder cancer and adjacent non-tumor tissue were detected by immunohistochemical staining.High staining rate of ZONAB protein in bladder cancer is higher than in adjacent non-tumor tissue,and high staining rate in muscle-invasive bladder cancer is higher than that in non-muscle-invasive bladder cancer.(4)Correlation between high staining rate of ZONAB protein and clinicopathological factors of bladder cancer patients: High staining rate of ZONAB protein expression was not related to clinicopathological factors as age,gender,lymph node metastasis and distant metastasis detected by immunohistochemical staining in bladder cancer patients.(5)ZONAB mRNA expression in each urothelial cell lines: ZONAB mRNA expression is higher in bladder cancer cell lines 5637,T24 and UM-UC-3 than that in immortalized human urothelial cell line sv-huc-1 detected by realtime-PCR.(6)ZONAB protein expression in each cell lines: ZONAB protein expression is higher in bladder cancer cell lines 5637,T24 and UM-UC-3 than in that in sv-huc-1 detected by Western Blotting.2.ZONAB overexpressing T24 cell line enhanced invading capacity(1)Construction of ZONAB overexpressing T24 cell line: Human bladder cancer cell line T24 was stably transduced by lentiviral vector.ZONAB expressing lentivirus was packaged with ZONAB vector pLV-ZONAB-DU.T24 cells were transduced with ZONAB expressing lentivirus.ZONAB overexpressing T24 cell line was screened.Stability overexpressing ZONAB protein in T24 cell line ZONAB(T24-ZONAB)was verified by Western blotting.(2)Detection of invasion capacity in each T24 cell line expressing different levels of ZONAB: Invasion rate of T24-P,T24-GFP and T24-ZONAB 3 cell lines were detected by Transwell chamber invasion assay.The invasion rate of T24-ZONAB group was significantly higher than control groups.3.ZONAB downregulation in UM-UC-3 cell line attenuates invasion.(1)Construction of ZONAB downregulated UM-UC-3 cell line: Human bladder cancer cell line UM-UC-3 was stably transduced using shRNA.ZONAB downregulated UM-UC-3 cell line was screened.Downregulation of ZONAB protein in UM-UC-3 cell line was verified by Western blotting.(2)Detection of invasion capacity in each UM-UC-3 cell line expressing different levels of ZONAB: Invasion rate of UM-UC-3-P(untransduced blank control),UM-UC-3-N(negative control)and UM-UC-3-R(test group)3 cell lines were detected by Transwell chamber invasion assay.The invasion rate of UM-UC-3-R group was significantly lower than control groups.4.The invasion promoting role of ZONAB expression is verified by in vivo study.(1)Xenograft animal model of bladder cancer:Tumor growth rate of T24-ZONAB group was significantly higher than that of T24-P group and T24-GFP group.Tumor size of T24-ZONAB group was significantly larger than that of the other two groups,indicating that ZONAB expression can promote proliferation of tumor cells.(2)Detection of bladder cancer metastasis and invasion in cancer cells with different ZONAB expression levels: T24-ZONAB bladder cancer xenograft animal model had obvious fluorescence at transplanted tumor site detected by small animal fluorescence imaging,which suggests that transplanted tumor site has a stable ZONAB expression,but no definite distant metastasis was found.No invasion into adjacent tissues was found in T24-P group and T24-GFP group,while invasion was found in multiple adjacent striated muscles in T24-ZONAB group,which suggests that ZONAB expression promotes tumor invasion.Conclusion:1.ZONAB expression is upregulated in bladder cancer.2.ZONAB expression is higher in muscle invasive bladder cancer than that in non-muscle invasive bladder cancer.3.ZONAB expression has an effect of promoting cancer invasion.