The Clinicobiological Significance And Molecular Mechanism Of The Aberrant Expression Of CYR61 Gene In Bladder Cancer

The biological characteristics, clinical treatment, prognosis are distinct for the two subtypes of bladder urothelial carcinoma, non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Accurate staging of the bladder cancer is pivotal in optimal therapy planning. However, it is insufficient for urologists in preoperative determining whether the tumor has invaded within the muscularis through cystoscope and imaging methods (CT or MRI). Searching ideal molecular classification biomarkers for bladder cancer is a hotspot in urological surgical oncology.Based on the mRNA expression profile derived from a microarray analysis with 16 NMIBCs and 14 MIBCs in previous study of this laboratory, data mining was performed to filter the differentially expressed genes between the two subtypes of bladder cancer. Eleven genes were selected (p<0.001 and fold change>2.0), and then validated by Realtime PCR applied in the tumor tissues, obtained in two bladder cancer sample sets (30 cases for the microarray screening set, and 54 cases for an independent set). Four significantly differential expression genes were found finally selected through the microarray screening and Realtime PCR validation. Among them, CYR61, the most significantly differential expression gene between the NMIBCs and MIBCs, was confirmed with the protein expression by immunohistochemistry staining in formalin fixed and paraffin embedded sections of the tumor tissue derived from 115 bladder cancer patients, and correlation between CYR61 expression and the disease-free survival (DFS) of the patients were also evaluated. The result showed higher expression of CYR61 in MIBCs than that in NIMBCs (p=0.001), but there was no correlation between CYR61 expression and the DFS of the patients (p=0.571). Urinary protein concentrations of CYR61 and other three reported biomarkers (MMP2, VEGF and VEGFC) were measured by ELISA in preoperative urine samples of 104 bladder cancer patients. CYR61 protein level in urinary supernatant was higher in MIBCs than that in NMIBCs, with a sensitivity of 72.7% and a specificity of 86.0% to differentiate MIBCs and NMIBCs; whilst the combination assay of CYR61+VEFGC+MMP2+VEGF could achieve a sensitivity of 87.9% specificity of 86.4%. The diagnostic accuracy of urinary CYR61 for identify MIBCs was higher than VEGFC, MMP2,VEGF, and also higher than imaging methods (CT or MRI).Experiments in vitro were also performed, to explain the function of CYR61 in bladder cancer, and to explore the mechanism of CYR61 overexpression in MIBC. First, higher expression of CYR61 was detected in invasive bladder cancer cell lines, comparing with non-invasive bladder cancer cells. Secondly, overexpression and knockdown approaches proved CYR61 could significantly affect the invasion and migration of the invasive bladder cancer cells but have no effect on non-invasive bladder cancer cells. It was also found that the expression change of CYR61 could significantly affect the gene expression of MMP2 and NRP1 in invasive bladder cancer cell lines but not in non-invasive bladder cancer cells. It was proved that the DNA copy number alteration was not the cause of higher expression of CYR61 in MIBCs. miR-30e, a significantly differential expressed miRNA between NMIBCs and MIBCs selected by miRNA expression microarray in previous study of this laboratory, was demonstrated, using dual luciferase report system, that the miRNA-30e it could combine with the 3’UTR of CYR61 and regulate the expression of the gene. It was also observed that the expression of miR-30e and CYR61 was negatively correlated, in the tumor tissue samples derived from 72 bladder cancer cases.In this study, based on high-throughput screening a panel of differentially expressed genes between NMIBCs and MIBCs were selected and validated. Among them, CYR61 was chosen for further investigation. The major novel findings of the study include that abnormal expression of CYR61 in bladder cancer is associated with the tumor invasion; and that urinary CYR61 protein is a promising biomarker for preoperative diagnosis of MIBCs.