Proteomic Analysis Of High Malignant Bladder Transitional Cell Carcinoma And Verification On Mortalin

ObjectiveBladder carcinoma is the most prevalent tumor of the urinary tract.In our country,the incidence rate and the case fatality of bladder carcinoma are highest in urinary system,so bladder cancer remains to be a focus in cancer research.More than 90%of bladder tumors are diagnosed as BTCC.Recent studies have found that,from biological behavior to pathological characteristics,BTCC actually has two phenotypes:low malignant and high malignant,so it can be called as two types bladder transitional cell carcinoma or two-tie grading system.This new classification provides the new molecular base for the study of bladder cancer.Further study may elucidate the molecular mechanism of bladder carcinogenesis and provide potential clinical diagnostic and therapeutic targets,and have important significance.On the basis of two-tie grading system,using a comparative proteomic approach,we found the differentially expressed proteins between high malignant bladder transitional cell carcinoma and normal bladder transitional epithelium.Furthermore,we verified the protein which might be a potential and valuable bladder biomarker and investigated its clinical value preliminary. Methods1.Firstly,the proteins of the high malignant bladder transitional cell carcinomas and normal tissue was separated by hand microdissection combining two-dimensional electrophoresis and Coomassie brilliant blue techniques.Then we screened the differentially expressed protein spots by image analysis software and artifical contrast,which relative value exceeded three.2.Using matrix-assisted laser desorption/ionization-time of flight-time of flight mass spectrometry(MALDI-TOF/TOF-MS) to identify the proteins which were separated by 2-DE,and the functions of proteins were searched through internet.3.The differentially expressed protein mortalin was verified by semi-quantitative RT-PCR and immunohistochemistry at mRNA level and protein level.Expanded immunohistochemistry was used to test the expression of mortalin in original bladder transitional cell carcinoma,and the correlation between immunohistochemistry results and clinicopathologic parameters was studied.Results1.We got the gel map of 2-DE successfully,and 20 protein spots,which expressed much more differentially,were screened by image analysis software.2.The above-mentioned 20 proteins were identified by MALDI-TOF/TOF-MS and the peptide mass spectrometry maps and amino acid sequences were obtained successfully,but combined with the database search,only 11 proteins were identified.Among all of these,CK7,Prohibitin,Hsp60,PDIA6,Tubulin beta-Ⅲ, mortalin,AnnexinA5 were up-regulated in high malignant BTCC,and CK8, 14-3-3 protein epsilon,Desmin and AnnexinA5 were down-regulated.We searched the proteins from PubMed and found 5 proteins had been reported that perhaps related with BTCC.There were little reports about the relationship between other 6 proteins with BTCC by now yet,including PDIA6,14-3-3εprotein,Desmin,Tubulin beta-Ill,Mortalin and Annexin A4.3.The screened protein mortalin was confirmed up-regulated in high malignant BTCC than that in normal tissues by semi-quantitative RT-PCR and immunohistochemistry,respectively.Expanded immunohistochemistry demonstrated that the expression of mortalin was weak in normal bladder transitional epithelium.Clinicopathological correlation analysis showed that mortalin overexpression in BTCC was significantly associated with pathological grades(R=0.601,P＜0.01) and advanced tumor TNM stages(R=0.021,P＜0.05). No apparent correlation with other clinical features,including tumor size,location, numbers,sex and age was observed.Conclusion1.The differentially expressed proteins between high malignant BTCC and normal bladder tissues were acquired by hand microdissection combined with 2-DE and MALDI-TOF/TOF-MS techniques.2.The protein profile of high malignant BTCC displayed obviously difference compared with normal bladder tissues.The results implied that various distinct proteins lead to bladder carcinogenesis. 3.The identified protein mortalin was up-regulated in high malignant bladder transitional cell carcinoma obviously.The results of expanded immunohistochemistry assay and correlation analysis of clinicopathologic data implied that mortalin might be a potential and valuable biomarker for BTCC.