The Study On Expression Of Mammalian Tissue Inhibitor Of Metalloproteinase Protein-4 In Bladder Carcinoma And Its Effect On The Proliferation,invasion And Metastasis Of Bladder Carcinoma Cells

Background:Bladder cancer is a kind of malignant lesions which has the highest incidence rate in the urinary system.In male,the incidence of bladder cancer is ranked the seventh in malignant tumor of the whole body,while in women it ranked tenth.Bladder cancer has a multicentered,easy-relapsing highly invasive,and drug-resistant biological feature.10% ~ 30% of NMIBC will develop into MIBC and high-level NMIBC are more likely to deteriorate.Nearly 75% of the high-level NMIBC patients will undergo recurrence,progression or death within 10 years after the diagnosis.There is no effective means and technique to accurately assess the prognosis of the tumor.In recent years,it is found that matrix metalloproteinases inhibitor-4(TIMP-4)has the ability of inhibiting tumor cell‘s proliferation and metastasis.TIMP-4 is one of the members of the family of TIMPs whichis the natural inhibitors of MMPs.TIMP-4 is expressed in a variety of malignant tumor tissues and cells,such as endometrial cancer,hepatocellular carcinoma,breast cancer,colon cancer,prostate cancer,lung cancer,glioma,and so on.Researches at home and abroad proved that it can regulate cell proliferation,apoptosis and the formation of tumor blood vessels.Increasing the TIMP-4 gene expression can effectively inhibit tumor cell proliferation,invasion and metastasis.But so far there is no reports about bladder cancer in regards of TIMP-4.Objective:1.To investigate the expression of mammalian tissue inhibitor of metalloproteinase protein-4(TIMP-4)in bladder urothelial cell carcinoma(BUCC)and normal tissue adjacent to carcinoma,to clear the relationship between the expression level and BUCC‘s histological stages.2.To prepare renal carcinoma cell line T24/TIMP-4 via plasmid transfection technique that could express TIMP-4 at a higher level,then investigate the change of bladder carcinoma cell‘s proliferation,invasion and migration ability.Methods:1.The study of TIMP-4 expression level on different pathological tissuesThe pathological tissue group of 152 cases that have been diagnosed as bladder carcinoma were collected.Bladder carcinoma tissues were classified as experimental group,and normal tissues adjacent to carcinoma were classified as control group.Paraffin sections that contain the two groups,then SP immunohistochemical staining was performed.Semi-quantitative analysis help us to get the difference of TIMP-4‘s expression level between the two groups,even more,the difference between different histological stages in BUCC.2.The biological behavior study of TIMP-4 on BUCCThe bladder carcinoma strain T24 transfected by TIMP-4 gene plasmid was experiment group,the T24 transfected by P-CMV4 plasmid was control group,and the T24 cell strain without anything was blank group.The TIMP-4 protein and mRNA expression level of each cell strain were detected by qRT-PCR and western-blot.The proliferation ability of each cell strain were detected by MTT assay and colony formation assay.The invasion and metastasis ability of each cell strain was analyzed by Transwell assay and Wound-healing assay.Results:1.The study of TIMP-4 expression level on different pathological tissuesThe rate of TIMP-4 positive expression in BUCC tissue specimens was 26.97%,which in normal tissue adjacent to carcinoma was 84.21%(P<0.05).In BUCC tissue,there has a different expression between the different histological stages.The rate of low-grade and high-grade was 38.71% and 18.89%,respectively.From the result of Chi-square test,the TIMP-4 expression level in high-grade was lower than low-grade.2.The biological behavior study of TIMP-4 on BUCCThe TIMP-4 protein and mRNA expression level of each cell strain were detected by qRT-PCR and western-blot.The result shows the experiment group T24/TIMP-4 cell protein expression and mRNA expression level of TIMP-4 are higher than the control group and blank group.The cell growth curves and colony formation showed that overexpression of TIMP-4 could suppress the growth of T24 cells more than that of the other two groups.Transwell migration assays was performed with T24 cells.The number of T24/TIMP-4 cell(6.67±2.11)that had through the membrane was lower than that in control group(15.43±4.22)and blank group(13.23±3.23)(P<0.01).Similarly,the migration distance of T24/TIMP-4 cell was shortest in Wound-healing assay(P<0.01).Conclusion:The TIMP-4 are expressed in BUCC and in normal tissue adjacent to carcinoma,but there has a different expression level.The expression level in BUCC was lower than the normal tissue adjacent to carcinoma.About BUCC,the level is connected with tumor‘s histological stages,in other words,that of high-grade are lower than that of low-grade.Through the plasmid transfection technique,we prepared BUCC cell lines that could overexpressed TIMP-4,meanwhile,that suppresses the bladder carcinoma cell proliferation,invasion and metastasis.