The Prelimilary Study Of The Clinical Significance And Biological Mechanism Of The γ-synuclein As A Molecular Marker Of The Bladder Carcinoma

Background:Bladder cancer is the most common malignant tumor of urinary tract that threat to human health, among which 90% or more are urothelial carcinoma. Among the patients who are initially diagnosed with bladder cancer, about 75% are non-muscle invasive bladder cancer, and the classical transurethral resection has a good effect for them. However,50% to 70% of them will relapse and 10 to 15% of the recurred tumor will progress to muscle invasive. So, a lifelong follow-up is necessary for the patients with bladder cancer.Cystoscopy and voided urine cytology (VUC) are still the gold standards for the diagnosis and follow-up of bladder cancer. However, as an invasive procedure, cystoscopy is not easily to be accepted by patients due to discomfort caused by the examination; while VUC has a low overall sensitivity with less than 40% for the low-grade tumors, as such, a negative result cannot exclude the presence of a low-grade disease. Commercial tests that detect Nuclear Matrix Protein 22 (NMP22), Bladder Tumor Antigen (BTA) and fibrin degradation products (FDP) are FDA-approved tests for bladder cancer diagnosis and there is higher sensitivity compared with VUC, but they are not widely used due to the low specificity. Therefore, it is urgent need to identify biomarkers with high sensitivity and specificity for early diagnosis and postoperative surveillance of bladder cancer.The synucleins families are a small, soluble, highly conserved group of neuronal proteins that mainly expressed in neural cells, the family consists of α-, β-, and y-synuclein. They are a natively unfolded group of proteins that share sequence homologies and structural properties. So far, the biological functions of the synucleins are still unclear, α-synuclein (SNCA) and β-synuclein (SCNB) mainly related to neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, while y-synucle (SNCG) mainly related to the neoplasm. Recently, elevated levels of SNCG proteins have been detected in various types of cancer, especially in advanced stages of the disease. And, it promotes cell proliferation, invasion and metastasis, also it interferes with drug-induced apoptotic responses. Currently, more and more attentions were paid to the association between SNCG and a wide range of cancer types such as breast, ovarian, liver, lung, gastric, esophagus, pancreatic, bladder and so on. It implies that SNCG maybe serve as an aggressive biologic marker and therapeutic target for cancer.Object:To preliminarily explore the clinical significance and biological mechanism of the SNCG in diagnosis and prognosis monitor of the bladder cancer. Provide a foundation for the further study and clinical appliance of the SNCG in bladder cancer.Methods:1.301 bladder cancer specimens and 98 corresponding non-neoplastic adjacent tissues with complete clinical and follow-up data were retrospectively collected. Immunohistochemical analysis was used to detect the SNCG expression, and then the associations between SNCG expression and clinicopathological characteristics and prognosis of bladder cancers were analyzed.2. ELISA method was performed to detect SNCG in the serum and urine in 58 patients with bladder cancer, and the differences of the SNCG expression between the serum and urine was compared.3. Western blot was performed to detect urine SNCG, SNCG short and long chain in corresponding tissues in 17 patients with bladder cancer, and the concentration of beta-actin in control group.4. Among 95 patients initially diagnosed with bladder cancer, the preoperative urine and the postoperative urine (before discharge,1 month later,3 months later and 6 months later, to end with recurrence). Then ELISA method was performed to detect the SNCG in each urine specimen, and the change of the SNCG was observed dynamically, at the same time, the associations between SNCG expression and clinicopathological characteristics was analyzed.5. RT-PCR, Western blot were performed to detect the expression of SNCG in bladder cancer cell lines EJ, T24,5637 and UMUC, and the cell line with the highest SNCG expression was screened.6. The SNCG RNAi sequences and control sequence were prepared and transfected into bladder cancer cell line 5637. The sequence with stably SNCG RNAi expression was screened.7. The proliferation capacity of 5637 cells with the SNCG down-regulated by RNAi was detected by CCK-8 method.8. Transwell Chambers was performed to evaluate the invasion capability of the 5637 cells with the SNCG suppressed by RNAi.Results:1. SNCG is highly expressed in bladder cancer tissues, and is realated to the recurrence of the bladder cancer. Kaplan-Meier curves estimation showed that the DFS and OS of the group with SNCG negative are longer compared with the group with SNCG positive; the DFS and OS of the SNCG-negative were (95.7±4.3) months (95%C7,87.3～104.1 months), (94.8±4.0) months (95%CI,87.0～102.6months), respectively, for the SNCG-positive, the DFS and OS were reduced to (71.5±4.9) months (95%CI,61.9～ 81.0months), (85.9±5.5) months (95%CI,75.1～96.6months), respectively (P=0.010 and 0.022, respectively); multivariate Cox analysis demonstrated that SNCG overexpression was an independent prognostic indicater for patients with bladder cancer (P=0.000).2. SNCG expression in the urine was significantly higher than that in serum, and the SNCG expressed in urine was highly consistent with that in the corresponding bladder cancer tissues;3. There is close relation between SNCG concentration in preoperative urine and the tumor infiltration depth. As to the same patient, the SNCG expression in postoperative urine declined obviously compared with that in preoperative urine. When bladder cancer recurred, the SNCG expression in urine rose again.4. The expression of SNCG in 5637 cell lines was detected higher than that in cell lines EJ,T24 and UMUC-3.5. The stable bladder cancer cell line 5637 with inhibition of the SNCG expression were successfully screened:siSNCG-244.6. The CCK-8 assay showed that inhibition of SNCG expression can reduce the proliferation capability of 5637 cell lines.7. The Transwell chamber demonstrated that SNCG-suppressed 5637 cell lines show a significant reduction in migration and invasion capacity.Conclusions:1. SNCG is highly expressed in bladder cancer tissues, and it can be regared as an independent prognostic indicater for bladder cancer, which suggested that the aberrant expression of SNCG may be associated with the occurrence and development of the bladder cancer.2. SNCG expression in urine was significantly higher than that in serum, and it was highly consistent with that in the corresponding bladder cancer tissues. This result suggested that bladder cancer cells could be directly secreted to the urine, and urine SNCG may be an ideal biomarker for the detection of bladder cancer.3. As to the same patient, the SNCG level in postoperative urine declined obviously compared with that in preoperative urine and when bladder cancer recurred, the SNCG level in urine rose again. This indicated that urine SNCG may be a biomarker for the recurrence of the bladder cancer.4. Down-regulation of SNCG in 5637 cell lines affected its malignant phenotype, including inhibiting cell proliferation, migration and invasion. siRNA-mediated SNCG down-regulation inhibited the malignant phenotype of 5637 cell lines, suggesting that SNCG may become a potentially therapeutic target for bladder cancer treatment.