monstrated by Western blot analysis. On the mRNA level quantitative PCR also revealed reduced amounts of FAIM molecules relative to human acidic ribosomal protein as a house keeping gene. MiR-133b impairs expression of glutathione-S-transferase pi Microarray analysis of miR-mediated gene regulation relies exclusively on mRNA decay. Since miR-mediated gene expression repression also occurs at the translational level, we decided to perform pulsed stable isotope labeling with amino acids in cell culture analysis of HeLa cells transfected with miR-133b or control miR. As done previously, transfected cells were stimulated with TNFa for 6 h prior to protein lysate harvest. In accordance with previous reports about quantitative effects of miR activity on the cellular proteome, ectopic introduction of miR133b into HeLa cells led only to mild effects on overall protein biosynthesis. In line with this, cross-comparison of pSILAC results with ” microarray expression analysis revealed repression of GSTP1 at the mRNA level. This had not been identified previously, because of the filtering strategy applied to the miR-133b target predictions according to miRecords. According to the in-silico prediction tools TargetScan and MicroCosm Targets, GSTP1 displays one single miR-133b binding site in its 39-UTR . As demonstrated by luciferase reporter assays, coupling of entire GSTP1 39-UTR to Renilla luciferase specifically rendered it less active after cotransfection of miR-133b. Supporting a direct ” interaction between miR-133b and the cloned target sequence, mutation of the predicted seed sequence reinstated enzymatic activity. Moreover, qPCR analysis corroborated the suppressive effect of miR-133b on the mRNA levels of GSTP1. As the final line of evidence, Western blot analysis confirmed the pSILAC results by proving protein levels of GSTP1 to be miR-133b sensitive in HeLA cells. Using pSILAC FAIM was not identified. MiR-133b is downregulated in prostatic tissue from cancer patients and induces proliferation arrest in PC3 cells Previous studies described the downregulation of miR-133b in cancer and discussed the potential of such miR signatures for diagnosis and prognosis. To prove the biological significance of miR-133b dysregulation in cancer, we examined miR-133b expression in 69 human prostate cancer samples and matched normal adjacent tissue. Although normalized ct-values varied in individual patients, mean miR-133b expression was 1.7-fold decreased in tumor tissue, with 75% of examined patients displaying miR-133b, a Potent Proapoptotic Molecule 1.0 0.8 0.6 0.4 0.2 0.0 detectable. Nevertheless, it did not seem to be induced. This points to an ambivalent function of miR-133b, which can be pro- or buy Indirubin-3′-oxime antiapoptotic depending on cell type. Discussion Herein, we used human cervix and prostate carcinoma cells as a model system for DR-triggered apoptosis to interrogate whether miR-133b is capable of modifying the cellular death response to TNFa, Fas/CD95 ligand or TRAIL. Our findings reveal that miR-133b is a potent catalyzer of DR-mediated cell death. The accentuated apoptotic response of miR-133b transfectants is a direct consequence of synergistic alterations of their protein repertoire. Specifically, miR-133b directly impairs the expression of important antiapoptotic genes. We provide evidence for FAIM as a target of miR-133b. At present, two major isoforms of FAIM have been described. The long isoform is present almost exclusively in neurons, whereas the sh
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