| Backgroud and objective:Urinary Bladder cancer, one of the most common malignant tumors, is the 11th most common cancer worldwide, accounting for 3% of the global cancer incidence. There were about 429,800 new cases of bladder cancer and 165,100 deaths in 2012 worldwide. The majority of bladder cancer occurs in men. Bladder cancer is 3 to 4 times more common in men than in women. Incidence rates are highest in Europe, Northern America, Western Asia, and Northern Africa, and lowest in Eastern, Middle, and Western Africa. Urinary Bladder cancer is the fourth most common cancer among men in America. An estimated 74000 new cases of bladder cancer and 16000 deaths occurred in 2015. Turkey has the highest mortality among men, where the estimated death rate in 2012 (12.8 per100,000) was 3 times as high as that in the United States (4.0) and 50% higher than the highest rates in Europe (8.3 in Latvia and 8.0 in Poland). Incidence rates have shown an increased trend from 1998 to 2008 in China, increased from 2.21% of the China cancer incidence in 1998 to 2.5% in 2008. Urinary Bladder cancer is the eighth most common cancer in China, accounting for 2.5% of cancer incidence in 2008.Bladder cancer is divided into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC).70%-80% cases present at first diagnosis as well differentiated or intermediate differentiation non-muscle-invasive bladder cancer[4]. After the first transurethral resection of bladder tumor(TURBT), the recurrence in first year is 15% to 61% and 31% to 78% of patients will relapse in 5 year. What’s more,10% cases will progress to muscle-invasive bladder cancer or metastasis disease. Especially to high-risk NMIBC,45% patients will progress to invasive disease within 5 year. Invasion and metastasis are the leading causes to the death of bladder cancer patients. A research in our country reported that the five-year survival rate of NMIBC is 91.9% for Ta-1,84.3% for T2,43.9% for T3 and 10.2% for T4 repectively in China. The foreign researchs reported that the 5- and 10-year recurrence-free survival for patients with organ-confined, lymph node-negative tumors was 92% and 86% for PO disease,91% and 89% for Pis,79% and 74% for Pa, and 83% and 78% for P1 tumors, respectively. The 5- and 10- year recurrence-free survival for Patients with muscle invasive, lymph node-negative tumors was 89% and 87% for T2,78% and 76% for T3a,62% and 61% for T3b,50% and 45% for T4 respectively. While, the 5- and 10-year recurrence-free survival for these patients with lymph node invation tumor was 35% and 34%. This diverse biologic behavior compels current guidelines to recommend intense follow-up and invasive treatment and incur substantial costs. Therefore, it is of great clinical significance to determine the recurrence and invasive potential of these tumors. Some scholars think that it is better to doradical cystectomy to get the best treatment for those who have high risk of invasion and metastasis NMIBC. While grade and T category are the best estimators of subsequent NMIBC behavior, for example, which are used as the important risk factors to predicte recurrence and progression in EORTC Risk Tables, they are relatively imprecise measures for an individual patient, In present, despite efforts to identify molecular markers to predicte recurrence and progression for bladder cancer, there are no desirable ones in clinical management of NMIBC.Therefore, we tend to find differentially expressed gene between NMIBC and MIBC through making a data mining and analysing the gene expression data of bladder cancer downloaded from GEO datasets. Besides, a restrospective study was performed with large number of clinicl samples in order to gain a preliminary understand of the potential biological mechanism of NMIBC. This study has vital practical and scientific significance in intensive study of the molecular mechanism, guiding individualized treatment and improving prognosis of NMIBC.Materials and Methods1. Research objects1.1 bladder cancer tissue specimensWe collectde bladder cancer tissue specimens from the third hospital affiliated southern medical university and the cancer center of Sun Yat-sen University from January 2007 to May 2014. The samples were divided into three group.59 transitional cell bladder cancers tissues and 21 adjacent normal tissues from the patients who underwent surgery were collected for RTQ-PCR analysis,10 bladder cancers tissues and 4 adjacent normal tissues were used for Western Blot analysis. In addition,209 paraffin-embedded samples of transitional cell bladder cancer and 12 specimens of adjacent normal bladder tissue were collected for immunohistochemical assay. All tumors were histologically and clinically diagnosed by the pathologyists in cancer center of Sun Yat-sen University.1.2 Gene expression dataMicroarray datase, GSE3167, GSE37317, GSE31684and GSE5287, were downloaded from the United States center for biotechnology information Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/gds/) database. Microarrays with 55 non-muscle-invasive bladder cancer and 132 muscle-invasive bladder cancer tissues were analysed to find differentially expressed genes.2 Research methods2.1 Bioinformatics analysis.Using R software package, Gene expression profiling data was re-summarized using the RMA method and Entrez gene-centric CDF files (instead of original Affymetrix CDF files), which filtered out non-specific probes on the GeneChips and merged multiple probe sets representing the same Entrez gene into one probe set. Significance analysis of microarray (SAM) was performed to identify differently expressed genes between non-muscle-invasive bladder cancer and muscle-invasive bladder cancer tissues. Delta was set to 2.25, and the threshold of FDR was set to 0.001. The differently expressed genes were further analyzed with GenCLiP software (http://ci.smu.edu.cn) to do GO analysis and pathway analysis.2.2 Quantitative Real-Time PCR (qRT-PCR) analysis.Quantitative Real-Time PCR was used to analysis CDH11 mRNA in 59 paired transitional cell bladder cancers and 21 adjacent normal tissues, which were randomly selected. TBP was amplified as an internal control. Relative quantification (2’△△Ct) method was used for calculating fold changes.2.3 Western Blotting analysisTotal proteins were extracted from 10 bladder cancers tissues, including 5 NMIBC and 5 MIBC, and 4 adjacent normal tissues, which were randomly selected. Western Blotting and semi-quantitative analysis was performed with housekeeping protein GAPDH used as a control.2.4 Immunohistochemistry assay209 paraffin-embedded samples of transitional cell bladder cancer and 12 specimens of adjacent normal bladder tissue were randomly selected to test the expression of CDH11 protein using SP method. In brief, the intensity of immunostaining was scored as negative (0), weak (1), medium (2) and strong (3). The extent of staining, defined as the percent of positive staining cells, was scored as 1 (<10%),2 (11-50%),3 (51-75%) and 4 (75%). An overall expression score, ranging from 0 to 7, was obtained by plus the score of intensity and that of extent. The definition of staining for CDH11 was as follows:positive, when tumor cells showed immunoreactivity with a membranous staining (overall score≥1); negative, when 0% tumor cells showed immunoreactivity (overall score=0).2.5 Transwell and Boyden assayTranswell migration assay and matrigel invasion assay were used to analysis the effect of CDH11 on the migration and invation of T24, BIU87, EJ and 5637 bladder cancer cells.2.6 Statistical analysesStatistical analyses were performed with the SPSS 20.0 statistical software package. Comparisons between two groups were performed using Student’s t-test, unless otherwise indicated. The association between CDH11 and clinical features was analyzed using chi-square text. Survival analysis and Univariate analysis was performed by Kaplan-Meier and log-rank test. Multivariate analysis was performed by Cox proportional hazard model. p< 0.05 was considered to be statistically significant.Result1. Bioinformatics analysis results showed genes which are related to EMT, such as CDH11, are contributes to the progression of NMIBC.SAM analysis identified 414 differentially expressed genes, in which 185 genes were up-regulated and the other 229 genes were down-regulated in muscle-invasive bladder cancer tissues. The genes were further analyzed with GenCLiP software (http://ci.smu.edu.cn) to annotate gene functions. GO analysis showed that those differentially expressed genes were major related to cell adhesion, extracellular matrix, cell proliferation, cell migration and so on. Pathway analysis showed that the potential pathway included integrin-linked kinase, transforming growth factor-P and so on. What’s more, there are 164 differentially expressed genes related to EMT. CDH11 is one of the biggest fold change of gene, amounting to 2.89 times, P＜0.0001.2. CDH11 RNA and protein was up-regulated in BC tissueQuantitative real-time PCR and Western Blot were used to examine RNA and protein level of CDH11 in BC tissues. CDH11 RNA and protein were found to be differentially overexpressed in human primary BC samples compared with non-cancerous samples. What’s more, the RNA and protein expression of CDH11 were significantly higher in muscle-invasive bladder cancer than non-muscle-invasive bladder cancer. Immunohistochemistry found that CDH11 protein didin’t expressed in normal bladder epithelium. Membranous CDH11 staining was detected at the cancer cell membrane and recorded in 30.5%(57/187) NMIBC and 54.5%(12/22) MIBC. Statistical analysis revealed a significant increased CDH11 expression in muscle-invasive carcinomas compared with superficial bladder tumors.3. Cadherin-11 increases migration and invasion of bladder cancer cellsQuantitative real-time PCR and Western Blot were used to examine RNA and protein level of CDH11 in four BC cell lines (5637,EJ,T24 and BIU87).We found that CDH11 was high expression in T24 and BIU87 and low expression in EJ and 5637. Transwell and Boyden assay found that CDH11 increased migration and invasion of bladder cancer cells.4. The significance of CDH11 expression in bladder cancerTo investigate the oncogenic role of CDH11 in BC, we analyzed the association between CDH11 and clinicopathologic indicators in BC patients. Statistical analyses indicated that the CDH11 expression was positively associated with grade, T category, relapse and progression. More importantly, patients with positive expressions of CDH11 were more vulnerable to relapse and progress than those with negative expression. Univatiate and multivariate analysis showed that CDH11 was a significant and independent prognostic predictor for progress-free survival and recurrence-free survival of patients with NMIBC.Conclusion1. Bioinformatics analysis results showed genes which contributes to EMT(such as CDH11) promote to the progression of bladder cancer after surgery.2. CDH11 RNA and protein was up-regulated in BC Tissue. CDH11 was expressed higher in NMIBC than that inMIBC.3. High expression of Cadherin-11 increased migration and invasion of bladder cancer cells.4. CDH11 expression was positively associated with grade, T category, relapse and poor progression. CDH11 may serve as an independent prognostic predictor for bladder cancer patients. |
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