Overexpression of miR-128 specifically inhibits the truncated isoform of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene <it>NTRK3 </it>yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of <it>NTRK3 </it>are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system.</p> <p>Results</p> <p>We identify one microRNA (miR-151-3p) that represses the full-length isoform of <it>NTRK3 </it>and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated <it>NTRK3</it>, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor <it>BCL2</it>.</p> <p>Conclusions</p> <p>Our results show that the regulation of <it>NTRK3 </it>by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.</p

Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

[Abstract] Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-0040Ministerio Ciencia e Innovacion; PLE2009-0144Ministerio Ciencia e Innovación; PI 08/202

Design and evaluation of a panel os single-nucleotide polymorphisms in microRNA genomic regions for association studies in human disease

MicroRNAs (miRNA) are recognized posttranscriptional gene repressors involved in the control of almost every biological process. Allelic variants in these regions may be an important source of phenotypic diversity and contribute to disease susceptibility. We analyzed the genomic organization of 325 human miRNAs (release 7.1, miRBase) to construct a panel of 768 single-nucleotide polymorphisms (SNPs) covering approximately 1 Mb of genomic DNA, including 131 isolated miRNAs (40%) and 194 miRNAs arranged in 48 miRNA clusters, as well as their 5-kb flanking regions. Of these miRNAs, 37% were inside known protein-coding genes, which were significantly associated with biological functions regarding neurological, psychological or nutritional disorders. SNP coverage analysis revealed a lower SNP density in miRNAs compared with the average of the genome, with only 24 SNPs located in the 325 miRNAs studied. Further genotyping of 340 unrelated Spanish individuals showed that more than half of the SNPs in miRNAs were either rare or monomorphic, in agreement with the reported selective constraint on human miRNAs. A comparison of the minor allele frequencies between Spanish and HapMap population samples confirmed the applicability of this SNP panel to the study of complex disorders among the Spanish population, and revealed two miRNA regions, hsa-mir-26a-2 in the CTDSP2 gene and hsa-mir-128-1 in the R3HDM1 gene, showing geographical allelic frequency variation among the four HapMap populations, probably because of differences in natural selection. The designed miRNA SNP panel could help to identify still hidden links between miRNAs and human disease.This work was supported by the 'Instituto Carlos III and Fondo de Investigaciones Sanitarias' [CIBER-CB06/02/0058, CIBER-SAM, FIS/ISCIII:P1052565, ISCIII:GO3/184], the 'Fundació la Marató-TV3' [014331], the 'Departament d’Universitats Innovació i Empresa, Generalitat de Catalunya' [2005SGR00008] and the European Union Sixth Framework Programme Integrated Project SIROCCO [Grant LSHG-CT-2006-037900