The Diagnostic And Biological Role Of Endocan In Bladder Cancer

Background Bladder cancer is a malignant disease with high morbidity and mortality all over the world.In China,it is the malignancy with the highest incidence in urinary system.According to the invasion depth into underlying tissue,bladder cancer can be classified into non-muscle invasive and musclce-invasive.These two types of bladder cancer are different in biological phenotypes,prognosis and treatments.Non-muscle invasive bladder cancer(NMIC),accounting for 75% of all bladder cancer,can have good prognosis if diagnosed and treated early.Nevertheless,it tends to recur.The standard treatment of NMIBC is transurethral resection of bladder tumor,supplemented with postoperative intravesical chemotherapy like mitomycin or BCG instillation to prevent tumor recurrence and progression.Musclce-invasive bladder cancer(MIBC)has poor prognosis with a high risk of metastasis.More progressive treatment such as radical cystectomy plus urinary diversion is necessary for MIBC.Radiotherapy or systemic chemotherapy should be considered according to the surgical margin,postoperative recurrence or distant metastasis.The heterogeneity of invasion depth and prognosis of bladder cancer results from the differences in genes and proteins of cancer cells.Studying the genetic differences and exploring the mechanism of progression of bladder cancer are prerequisites for early diagnosis and precision treatment.The established diagnostic techniques for bladder cancer are urinary cytological pathology and cystoscopy.Urinary cytology is a non-invasive diagnostic technique with high specificity but low overall sensitivity(less than 20% for low-grade bladder cancer).Cystoscopy is the bladder endoscope inserted into the bladder through the urethra,examined by naked eyes of urologists.If a suspicious neoplasm is detected,tissue clamp is used for biopsy.Cystoscopy is an invasive procedure that can result in damage to the urethra and bladder mucosa,causing bleeding and even urinary infection.Moreover,cystoscopy is painful and psychologically discomfortatable,resulting in low patient compliance.Given the shortcomings of current diagnosis of bladder cancer,urine-based non-invasive diagnostic markers have clinical significance.The development of next-generation sequencing and bioinformatics provide resources for protential bladder cancer diagnostic and subtyping markers.And it also provides powerful tools and clues to understand the mechanism of bladder cancer progression.For better understanding of the genome of bladder cancer in China,and for potential diagnostic markers,we collected bladder cancer samples and urine samples from different patients.Using genetic sequencing,in vitro and in vivo molecular experiments,we explore diagnostic markers and the molecular mechanism of bladder cancer progression.Methods 1.Transcriptome sequencing and bioinformatics analysis were used to sequence and analyze the paired bladder cancer samples and normal urothelial samples to find differentially expressed genes and protential biomarkers.2.Total RNA was extracted from tissue samples for quantitative PCR to verify Endocan expression results from sequencing.3.We retrieved bioinformatics platform Oncomine to analyze Endocan in other bladder cancer studies.4.Urine was collected from bladder cancer patients and control group.Enzyme-linked immunosorbent assay(ELISA)was used to determine urinary Endocan.And results were normalized by urine creatinine concentration.5.We produced tissue microarrays using bladder cancer and normal urothelial samples.The expression of Endocan microarray was determined by immunohistochemistry.6.Endocan was interfered by siRNA in T24 and T921 bladder cancer cell lines to study its role in tumor biological phenotypes such as proliferation,migration and invasion.7.Lentivirus was used to establish Endocan-knockdown bladder cancer cells that were implanted in nude mice for in vivo experiment.8.Flow cytometry was employed to investigate the effects of Endocan knockdown on bladder cancer cell cycle and apoptosis.9.The role of Endocan in bladder cancer cell autophagy was investigated by Western-blotting to analyze molecular mechnism.Results 1.In 10 paired tissue samples,Endocan was over-expressed in bladder cancer samples but not in normal mucosal tissues.2.In verifying phase,Endocan expression in bladder cancer was significantly higher than normal urothelial samples,and the expression level of Endocan was related to patholigical results.3.Endocan is over-expressed in bladder cancer in study from Oncomine databases.4.The normalized median endocan concentration was 6.08±0.75 in 92 bladder cancer patients,compared with 0.75±0.18 in the urine of 39 controls.And urinary Endocan was related to cancer pathology.Endocan has a potential for non-invasive diagnosis of bladder cancer.5.In tissue microarray,Endocan was significantly higher in bladder cancer than normal urothelial samples.And within bladder cancer samples,Endocan expression was higher in muscle-invasive bladder cancer than in non-muscle-invasive one.6.Endocan knockdown suppressed proliferation,migration and invasion in T24 and T921 bladder cancer cell lines.7.In in vivo experiments,Endocan knockdown restrained tumor growth in terms of tumor proliferation curve and endpoint tumor size.8.Interfering Endocan with siRNA can suppress cell apoptosis in T24 and T921 cells.No significant changes were found in cell cycle.9.Endocan knockdown can up-regulate bladder cancer cell autophagy.This effect may be mediated through mTOR.ConclusionEndocan is over-expressed in bladder cancer,especially in muscle-invasive one.Urinary Endocan has a potential for non-invasive bladder cancer diagnosis.The knockdown of Endocan can suppress bladder cancer proliferation,invasion and cell apoptosis.Endocan knockdown may play its role through up-regulation of autophagy,which is mediated by mTOR.