Identification Of Related Proteins By Proteomics In Urinary Bladder Carcinoma

Bladder cancer is the most common malignant tumor of urinary system in our country, which has the characteristics of high recurrence rate and increased staging after recurrence. More than70%of the patients under cystoscope have a recurrence or metastasis within2years after resection of the tumor, and the clinical stage and pathological grade progress. Therefore, in clinical patients of postoperative bladder cancer need closely follow-up and adjuvant therapy. While, there is still lack of sensitive specific bladder cancer molecular markers and therapeutic targets. It is important for the prognosis of bladder cancer patients to further screening and identification of new bladder cancer diagnosis and monitoring of molecular markers and therapeutic targets, establishing a monitoring system of predicting prognosis of bladder cancer and tumor molecular markers. Part I CRABP2and FABP5as novel potential biomarkers of urinary bladder carcinoma identified by proteomic profilingObjectives:There is still lack of sensitive specific bladder cancer molecular markers for diagnosis and postoperative follow-up monitoring. Technology of proteomics is the popular and effective method for identifying and screening tumor molecular markers.This study aims to use2D-DIGE and MALDI-TOF/TOF MS to screen and identify sensitive specific bladder cancer molecular markers for people.Materials and methods:Randomly selected3cases of tumor tissue specimens of bladder urothelial carcinoma in pathological diagnosis and paired specimens of normal bladder mucosa adjacent to carcinoma. Extracted tissue protein with Cy2, Cy3and Cy5fluorescent tags followed with2D-DIGE, obtained two-dimensional protein point mapping for variance analysis. Selected the significant difference of total protein point in the3cases for MALDI-TOF/TOF MS analysis,screening of new potential bladder cancer molecular markers.Using Western Blot to validation and analysis in32cases of tumor tissue specimens of bladder urothelial carcinoma.Results:Analysis the variance of protein atlas getting from2D-DIGE,significant differences of expression in71protein spots were found in3cases of bladder urothelial carcinoma tumor tissues and paired normal mucosa tissues adjacent to carcinoma,Among them63protein points were successfully analysised identified by MALDI-TOF/TOF MS. we found high expression in21proteins, There are two points identified as CRABP2and FABP5. Through analysis in32cases of bladder cancer tissue samples,we found that there were abnormal high expression in tumor samples of CRABP2and FABP5.Conclusions:CRABP2and FABP5have abnormally high expression in bladder cancer, there are new potential tumor markers of bladder cancer. Part II Anti-tumor efficacy of mitofusin-2in urinary bladder carcinomaObjectives:Mitofusion2(Mfn2) is a novel gene characterised as a cell proliferation inhibitor. Mfn2protein over-expression, mediated by an adenovirus, has a significant anti-tumor effect in A548and HT-29cells. However, there is no report on the effect of Mfn2on urinary bladder carcinoma (UBCC). Here, we sought to investigate the function of Mfn2in UBCC.Materials and methods:Mfn2expression in36paired UBCC samples was investigated by reverse transcription-polymerase chain reaction and Western blot analyses. An adenovirus encoding the complete Mfn2open reading frame (Ad-Mfn2) was used to infect UBCC cells, and an adenoviral vector encoding green fluorescent protein (Ad-GFP) was used as a control. The effects of Mfn2on cell-cycle distribution and apoptosis were assessed by flow cytometry and Western blot analyses.Results:The Mfn2protein showed significantly lower expression in UBCC tissues than nearby nontumorous tissues. Ad-Mfn2exhibited a significant anti-tumor effect in T24and5637cells. Mfn2overexpression in T24cells significantly inhibited cell proliferation, by arresting the transition of the cell cycle from the Gl to S phase, and induced apoptosis by upregulating active caspase-3and cleaved PARP levels. Mfn2also induced increased p21and p27expression levels, but down-regulated PCNA levels.Conclusions:Mfn2is a potential UBCC tumor suppressor gene, which showed significantly lower expression in tumor tissues than adjacent nontumorous tissues and could promote apoptosis and inhibit the proliferation of UBCC cells. Mfn2may become an important therapeutic target for treating UBCC.