Real-time TaqMan PCR analysis of immune-related genes in bladder cancer

A47

The pathogenesis of bladder cancer involves genetic alterations and various gene expression changes. In an effort to provide insight into the underlying molecular and genetic events, the mRNA expressions of 93 genes were analyzed using TaqMan^® Low Density Array technology (Applied Biosystems, USA). In the array, real-time quantitative RT-PCR was performed for a panel of immune-related genes simultaneously in a 384 well format on RNA obtained from mouse bladder tumors and normal mouse bladders.
 Bladder cancer was induced in C57BL/6 female mice (n=20) when they were 4-6 weeks old using poly-L-lysine (PLL) followed by a modified murine bladder tumor cell-line (MB49 secreting prostate specific antigen (PSA) as a reporter gene). Successful implantation of the mice was confirmed by measuring PSA in the urine using ELISA. Control mice (n=20) were instilled with PLL but not tumor cells. The mice were terminated 7 and 28 days after tumor implantation, and the bladders and draining lymph nodes were harvested for RNA analysis. One sample from each group was used for the LDA study while the rest of the samples were used in real-time PCR for validation. The accuracy of the array is between 60-70%.
 Gene expression analysis revealed distinct profiles in the immune-related genes. Only genes displaying at least a 2-fold difference in expression level from normal tissue were considered for analysis. 7 days after tumor implantation, 58 genes were up-regulated in the bladder. Of which, 12 genes were up-regulated more than 20 times compared to a normal bladder. Most of these genes (CCL5/RANTES, STAT1, CD3, CCR7, IL2Rα/CD25) are Th1 related genes involved in T cell activation. Others are pro-inflammatory, immune cell recruitment and apoptosis related. On the other hand, only 9 genes were up-regulated in the draining lymph node. Some of the up-regulated cytokine genes (IL4, IL5) are Th2-related and involved in B cells activation. 28 days after tumor implantation, 19 genes were up-regulated whilst 32 genes were down-regulated in the bladder. The up-regulated genes are also involved in immune regulation/recruitment and inflammation (GMCSF, IFNγ, CCL2, CXCL11/ITAC, IL1β). Interestingly, CTLA4 which is inhibitory to T cells is also up-regulated. The genes that are down-regulated are primarily endothelial receptors and structural component of basement membranes (collagen, fibronectin, endothelin, p-selectin, angiotensin II receptor) and apoptosis related genes (Bax, Fas, Bcl2, Bcl2-like1). Contrastingly in the draining lymph nodes, over 80 genes are down-regulated at day 28 indicating a shut-down of the immune response.
 In this study, we applied a high-throughput real-time quantitative RT-PCR assay to characterize changes associated with bladder cancer. This can help to better understand the biology associated with tumorigenesis and identify possible molecular targets. The challenge now is to evaluate the importance of these targets in tumor progression and immune evasion.