Oncoprotein 18 is necessary for malignant cell proliferation in bladder cancer cells and serves as a G3-specific non-invasive diagnostic marker candidate in urinary RNA

Abstract

Background Urine-based diagnostics indicated involvement of oncoprotein 18 (OP18) in bladder cancer. In cell culture models we investigated the role of OP18 for malignant cell growth. Methods We analyzed 113 urine samples and investigated two human BCa cell lines as a dual model: RT-4 and ECV-304, which represented differentiated (G1) and poorly differentiated (G3) BCa. We designed specific siRNA for down-regulation of OP18 in both cell lines. Phenotypes were characterized by cell viability, proliferation, and expression of apoptosisrelated genes. Besides, sensitivity to cisplatin treatment was evaluated. Results Analysis of urine samples from patients with urothelial BCa revealed a significant correlation of the RNA-ratio OP18:uroplakin 1A with bladder cancer. High urinary ratios were mainly found in moderately to poorly differentiated tumors (grade G2-3) that were muscle invasive (stage T2-3), whereas samples from patients with more differentiated non-invasive BCa (G1) showed low OP18:UPK1A RNA ratios. Down-regulation of OP18 expression in ECV-304 shifted its phenotype towards G1 state. Further, OP18-directed siRNA induced apoptosis and increased chemo-sensitivity to cisplatin. Conclusions This study provides conclusive experimental evidence for the link between OP18-derived RNA as a diagnostic marker for molecular staging of BCa in non-invasive urine-based

Sampling, Logistics, and Analytics of Urine for RT-qPCR-based Diagnostics

“…It rather describes the feasibility of this approach. This methodology has been applied in many studies describing RNA-based tumor makers, including microRNA and RNA polymerase II transcripts in human cells [21][22][23][24][25]. In the light of these successful applications of the protocol, this work describes a robust and quantitative technology for RNA-based marker detection in urine samples for diagnostic purposes.…”

Transcriptome analyses of urine RNA reveal tumor markers for human bladder cancer: validated amplicons for RT-qPCR-based detection

“…Thus, we developed and thoroughly characterised marker-specific pairs of PCR primers for PCR reactions for selected and promising markers described in this study. Because of very limited RNA amounts in human urine and the lack of sufficient patient material, we performed initial studies in the use of a cell culture model system which we recently described [ 32 ]. In this dual model, the human control group C is represented by RT-4 cells which were established from a G1 stage urinary bladder carcinoma [ 33 ].…”

“…This includes the collection of urine samples at small volumes, the stabilization of RNA, shipping, storage, and the quantitative detection of RNA transcripts via RT-qPCR. All steps are based on established standard operation procedures [ 21 , 32 , 36 ]. While the proof-of-concept has been reported manifold in the past including potential bladder cancer-specific tumor markers [ 19 , 37 , 38 ], it remains open, what kind of gold standard in this methodological category will be identified and whether RNA markers can support or substitute the current gold standard, i.e.…”