Targeting MicroRNA-143 Leads to Inhibition of Glioblastoma Tumor Progression.

Glioblastoma (GBM) is the most common and aggressive of all brain tumors, with a median survival of only 14 months after initial diagnosis. Novel therapeutic approaches are an unmet need for GBM treatment. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Several dysregulated miRNAs have been identified in all cancer types including GBM. In this study, we aimed to uncover the role of miR-143 in GBM cell lines, patient samples, and mouse models. Quantitative real-time RT-PCR of RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples showed that the relative expression of miR-143 was higher in GBM patients compared to control individuals. Transient transfection of GBM cells with a miR-143 oligonucleotide inhibitor (miR-143-inh) resulted in reduced cell proliferation, increased apoptosis, and cell cycle arrest. SLC30A8, a glucose metabolism-related protein, was identified as a direct target of miR-143 in GBM cells. Moreover, multiple injections of GBM tumor-bearing mice with a miR-143-inh-liposomal formulation significantly reduced tumor growth compared to control mice. The reduced in vitro cell growth and in vivo tumor growth following miRNA-143 inhibition suggests that miR-143 is a potential therapeutic target for GBM therapy.

MicroRNA-27a distinguishes glioblastoma multiforme from diffuse and anaplastic astrocytomas and has prognostic value.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that bind to 3'-untranslated (UTR) regions of target messenger RNAs to regulate protein synthesis. Reports have suggested that a set of specific miRNAs may be used as diagnostic and/or prognostic markers for astrocytoma grading. However, there are few studies of the specific miRNAs differentially expressed in each astrocytoma grade. MiRNA-containing total RNA was isolated from archived formalin-fixed, paraffin-embedded (FFPE) samples from WHO grade II-IV astrocytoma patients. The RNA was labeled and hybridized to Affymetrix miRNA 2.0 arrays. Statistical analysis identified several miRNAs differentially expressed in each astrocytoma grade. In particular, miR-27a, miR-210, and miR-1225-5p expression levels were able to differentiate grade IV from grade II and III astrocytomas as confirmed by real-time PCR. Kaplan-Meier survival analysis showed that disease progression occurred faster for Glioblastoma Multiforme (GBM) patients with a lower miR-27a expression level. Transfection of CRL-1690 GBM human cancer cells with a miR-27a oligonucleotide inhibitor followed by Real-time PCR identified six potential miR-27a target genes. Furthermore, the miR-27a oligonucleotide inhibitor induced CRL-1690 cell apoptosis. Taken together, our results provide additional miRNA signatures for distinguishing GBM from lower astrocytoma grades and suggest miR-27a as a prognostic and therapeutic target for GBM.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.