miR-193b is an epigenetically regulated putative tumor suppressor in prostate cancer.

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in 6 prostate cancer cell lines and nonmalignant prostate epithelial cells. Thirty-eight miRNAs showed increased expression in any prostate cancer cell line after 5-aza-2'-deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR-193b was methylated in 22Rv1 cell line at a CpG island approximately 1 kb upstream of the miRNA locus. Expressing miR-193b in 22Rv1 cells using pre-miR-193b oligonucleotides caused a significant growth reduction (p < 0.001) resulting from a decrease of cells in S-phase of the cell cycle (p < 0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR-193b-expressing 22Rv1 cells (p < 0.01). Altogether, our data suggest that miR-193b is an epigenetically silenced putative tumor suppressor in prostate cancer.

Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface-bilayers.

Profiling ion flux through human intracellular chloride ion channels using live-cell based techniques, such as patch-clamp electrophysiology, is laborious and time-consuming. The integration of scalable microfluidic systems with automatable protocols based on droplet-interface-bilayers (DIBs) within which ion channels are incorporated circumvents several limitations associated with live-cell measurements and facilitates testing in controllable in vitro conditions. Here, we have designed and tested novel microfluidic layouts for the formation of arrays of DIBs in parallel and developed the first example of a miniaturised, DIB-based, fluorescence assays for Cl- fluxing, allowing the investigation of the functional properties of the human chloride intracellular ion channel 1 (CLIC1). The microfluidic protocols relied on passive geometries for droplet pairing and DIB formation. Using recombinantly expressed CLIC1, we identified the best conditions to maximise protein integration into a lipid bilayer and the oligomerisation of the protein into functional ion channels. Finally, CLIC1 ion channel functionality was assessed relative to α-Haemolysin into microfluidic DIBs using the same Cl- fluxing assay.

Increased freedom of movement in the nascent chain results in dynamic changes in the structure of the SecM arrest motif.

Ribosomes are responsible for the synthesis of all cellular proteins. Due to the diversity of sequence and properties, it was initially believed that translating nascent chains would travel unhindered through the ribosome exit tunnel, however a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest, Escherichia coli (E. coli) secretion monitor (SecM) is one such stalling peptide. How and why these peptides interact with the exit tunnel is not fully understood, however key features required for stalling appear to be an essential peptide arrest motif at the C-terminus and compaction of the nascent chain within the exit tunnel upon stalling. Mutagenesis of the SecM arrest sequence has identified three conservative point mutations that can retain a degree of stalling in this highly conserved sequence. This level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A. Further analysis of these mutants by pegylation assays indicates that this increase in stalling activity during translation is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel possibly due to the improved flexibility of the nascent chain provided by the removal of a restrictive proline residue. The data presented here suggest that arrest sequences may be more prevalent and less highly conserved than previously thought, and highlight the significance of the interactions between the nascent chain and the exit tunnel to affecting translation arrest.

Association of SPINK1 expression and TMPRSS2:ERG fusion with prognosis in endocrine-treated prostate cancer.

PURPOSE: The aim of the study was to examine whether TMPRSS2:ERG fusion or SPINK1 protein expression is associated with hormone responsiveness of prostate cancer and can thus be used as a biomarker. EXPERIMENTAL DESIGN: Diagnostic needle biopsies from prostate cancer patients primarily treated by endocrine therapy were evaluated for TMPRSS2:ERG fusion with fluorescence in situ hybridization and SPINK1 protein expression with immunohistochemistry. RESULTS: The frequency of TMPRSS2:ERG fusion in 178 biopsies of hormonally treated patients was 34%. Of the fusion-positive cases, 71% showed deletion between the two genes, and 23% showed gain of the fusion. The fusion was associated with high Ki-67 staining (P=0.001), age at diagnosis (P=0.024), and tumor area (P=0.006), but not with Gleason score, T stage, M stage, prostate-specific antigen (PSA), or progression-free survival. Strong positive SPINK1 expression was found in 11% (21 of 186) of the biopsies. SPINK1-positive cases had significantly shorter progression-free survival compared with SPINK1-negative cases (P=0.001). The expression was not associated with any other clinicopathologic variables studied. In a multivariate analysis, SPINK1 expression showed independent prognostic value, with a relative risk of 2.3 (95% confidence interval, 1.1-4.6). SPINK1 expression and the fusion were not associated with each other. CONCLUSIONS: There was no association between TMPRSS2:ERG fusion and prognosis, suggesting that TMPRSS2:ERG rearrangement does not implicate hormone dependence of the cancer. SPINK1 expression, found in approximately 10% of prostate cancers, was associated with aggressive form of the disease and could serve as a biomarker in endocrine-treated prostate cancer.