Reintroduction of bladder cancer-downregulated miR-145 results in massive cell death of bladder cancer cell lines
Abstract:Small non-coding microRNAs (miRNAs) have recently emerged as central regulators of gene expression. Accordingly, miRNAs have been shown to regulate processes such as tumorigenesis, development, and differentiation. We have profiled the expression of 315 unique human miRNAs in clinical samples of normal urothelium and different stages of bladder cancer (Ta, T1, T2-T4). The expression profile of the miRNAs was examined by array analysis using locked nucleic acid (LNA)-modified probes and verified by quantitative real time PCR (qPCR). A range of miRNAs were found to be dysregulated in bladder cancer, and associated with progression of disease from superficial tumors to muscle-invasive tumors.
One of the top dysregulated miRNAs was miR-145, a commonly lost miRNA in cancer. In situ hybridization using LNA probes located miR-145 to the urothelium of normal bladder biopsies. In order to examine the effect of miR-145 loss, precursor miR-145 molecules were re-introduced into miR-145 deficient immortalized (HU609) and cancerous bladder cell lines (T24 and SW780). Re-introduction of miR-145 induced growth arrest and massive cell death in bladder cancer cell lines, that was dose- and time-dependent as determined by MTT reduction assay, release of LDH, nuclear condensation by Hoechst staining, and caspase activation. Interestingly, knockdown of exogenous miR-145 by transfection of miR-145 LNA molecules inhibited the cell death, whereas the pan-caspase inhibitor zVAD-fmk did not. Exon microarray analysis of miR-145-transfected cells has been performed to identify transcripts that were differentially expressed prior to the onset of cell death. Thus, miR-145 may be a novel tumor suppressor in bladder cancer that upon re-introduction in vitro activates a cell death programme via repression of its target transcripts.
Document Type: Abstract
Publication date: May 1, 2008