169,744 research outputs found
Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells
No full textDNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway
Human cardiac microtissues capture aberrant RNA splicing in doxorubicin-induced cardiotoxicity
No full textDifferent atrophy-hypertrophy transcription pathways in muscles affected by severe and mild spinal muscular atrophy
No full textBACKGROUND: Spinal muscular atrophy (SMA) is a neurodegenerative disorder associated with mutations of the survival motor neuron gene SMN and is characterized by muscle weakness and atrophy caused by degeneration of spinal motor neurons. SMN has a role in neurons but its deficiency may have a direct effect on muscle tissue.
METHODS: We applied microarray and quantitative real-time PCR to study at transcriptional level the effects of a defective SMN gene in skeletal muscles affected by the two forms of SMA: the most severe type I and the mild type III.
RESULTS: The two forms of SMA generated distinct expression signatures: the SMA III muscle transcriptome is close to that found under normal conditions, whereas in SMA I there is strong alteration of gene expression. Genes implicated in signal transduction were up-regulated in SMA III whereas those of energy metabolism and muscle contraction were consistently down-regulated in SMA I. The expression pattern of gene networks involved in atrophy signaling was completed by qRT-PCR, showing that specific pathways are involved, namely IGF/PI3K/Akt, TNF-alpha/p38 MAPK and Ras/ERK pathways.
CONCLUSION: Our study suggests a different picture of atrophy pathways in each of the two forms of SMA. In particular, p38 may be the regulator of protein synthesis in SMA I. The SMA III profile appears as the result of the concurrent presence of atrophic and hypertrophic fibers. This more favorable condition might be due to the over-expression of MTOR that, given its role in the activation of protein synthesis, could lead to compensatory hypertrophy in SMA III muscle fibers
Serum mirna profiling for early pdac diagnosis and prognosis: A retrospective study
No full textBackground: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. Methods: Serum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs expression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. Results: 2549 human miRNAs were screened out. At SAM, 76 differentially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large majority (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was associated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signalling pathways. Conclusions: A series of serum miRNAs was identified as potentially useful for the early diagnosis of PDAC, and for establishing a prognosis
Combinatorial cis-acting elements regulate tissue-specific expression of the cardiac troponin I gene in vitro and in vivo
No full textThe cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (-230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the -230/+126 base pair promoter express beta-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome
Circulating miR-26a as Potential Prognostic Biomarkers in Pediatric Rhabdomyosarcoma
No full textRhabdomyosarcoma (RMS) arises from myogenic precursors that fail to complete muscle differentiation and represents the most frequent soft tissue sarcoma in children. Two major histological subtypes are recognized: alveolar RMS, characterized by a more aggressive behavior and a greater proneness to metastasis, and embryonal RMS which accounts for the 80% of cases and carries a better prognosis. Despite the survival of patients with localized tumors has progressively improved, RMS remains a challenging disease especially for metastatic patients and in case of progressive or recurrent disease after front-line therapy. MicroRNAs, a class of small non-coding RNA, have emerged as crucial players in cancer development and progression, and their detection in plasma (circulating miRNAs) represents a promising minimally invasive approach that deserve to be exploited in clinical practice. We evaluated the utility of circulating miRNAs as diagnostic and prognostic biomarkers in children with RMS profiling miRNAs from plasma of a small cohort of RMS patients and healthy donors (HD) using a qPCR Cancer Panel. An assessment of hemolysis status of plasma using miR-451/miR-23a ratio was performed as pre-analytical analysis. Statistical analysis revealed that miRNAs expression pattern clearly distinguished RMS patients from HD (p < 0.05). Interestingly, plasma levels of muscle-specific miR-206 were found to be significantly increased in RMS patients compared to HD, whereas levels of three potential tumor-suppressor miRNAs, miR-26a and miR-30b/30c, were found lower. Reduced levels of circulating miR-26a and miR-30b/c were further measured in an independent larger cohort of patients (validation set) by digital droplet PCR. In particular, we evidenced that miR-26a absolute plasma levels were associated with fusion status and adverse outcome (p < 0.05). Taken together, these findings demonstrate the potential of circulating miRNA as diagnostic and prognostic biomarker in children affected by this malignancy and enforced the key role of miR-26a in pediatric rhabdomyosarcoma
Cardiac and smooth muscle cell contribution to the formation of the mouse pulmonary veins
No full textPrevious studies have demonstrated that the primordial pulmonary veins originate as an outgrowth of the atrial cells and anastomosis with the pulmonary venous plexus. As a consequence of this embryologic origin the tunica media of these vessels is composed of cardiac cells that express atrial specific markers (Lyons et al. [1990] J Cell Biol 111:2427-2436; Jones et al. [1994] Dev Dyn 200:117-128). We used transgenic mice for the cardiac troponin I (cTNI) gene and smooth muscle (SM) myosin heavy chain as differentiation markers, to analyze how cardiac and SM cells contribute to the formation and structural remodeling of the pulmonary veins during development. We show here that the tunica media of the adult mouse pulmonary veins contains an outer layer of cardiac cells and an intermediate SM cell compartment lining down on the inner endothelium. This structural organization is well expressed in the intrapulmonary veins from the beginning of vasculogenesis, with cardiac cells accumulating over preexisting roots of endothelial and SM cells and extending to the third bifurcation of the pulmonary branches without reaching the more distal tips of the vessels. On the other hand, SM cells, which are widely distributed in the intrapulmonary veins from the embryonic stage E16, accumulate also in the extrapulmonary branches and reach the posterior wall of the left atrium, including the orifices of the pulmonary veins. This event takes place around birth when the pulmonary blood flow starts to function properly. A model for the development of the pulmonary veins is presented, based upon our analysis
Transcriptome-wide selection and validation of a solid set of reference genes for gene expression studies in the cephalopod mollusk Octopus vulgaris
No full textOctopus vulgaris is a cephalopod mollusk and an active marine predator that has been at the center of a number of studies focused on the understanding of neural and biological plasticity. Studies on the machinery involved in e.g., learning and memory, regeneration, and neuromodulation are required to shed light on the conserved and/or unique mechanisms that these animals have evolved. Analysis of gene expression is one of the most essential means to expand our understanding of biological machinery, and the selection of an appropriate set of reference genes is the prerequisite for the quantitative real-time polymerase chain reaction (qRT-PCR). Here we selected 77 candidate reference genes (RGs) from a pool of stable and relatively high-expressed transcripts identified from the full-length transcriptome of O. vulgaris, and we evaluated their expression stabilities in different tissues through geNorm, NormFinder, Bestkeeper, Delta-CT method, and RefFinder. Although various algorithms provided different assemblages of the most stable reference genes for the different kinds of tissues tested here, a comprehensive ranking revealed RGs specific to the nervous system (Ov-RNF7 and Ov-RIOK2) and Ov-EIF2A and Ov-CUL1 across all considered tissues. Furthermore, we validated RGs by assessing the expression profiles of nine target genes (Ov-Naa15, Ov-Ltv1, Ov-CG9286, Ov-EIF3M, Ov-NOB1, Ov-CSDE1, Ov-Abi2, Ov-Homer2, and Ov-Snx20) in different areas of the octopus nervous system (gastric ganglion, as control). Our study allowed us to identify the most extensive set of stable reference genes currently available for the nervous system and appendages of adult O. vulgaris
Expression profiling characterization of laminin alpha-2 positive MDC
No full textIn the Caucasian population, patients affected by the most frequent forms of congenital muscular dystrophies (MDC) are commonly divided into two groups. The first is characterized by mutations of the gene for the laminin alpha-2 (LAMA2). The second is positive for this protein, highly heterogeneous, and has no specific genetic defect associated yet. We studied the skeletal muscle transcriptome of four LAMA2 deficient and six LAMA2 positive MDC patients by cDNA microarrays. The expression profiling defined two patients groups: one mild and one severe phenotype. This result was in agreement with histopathological features but only partially with the clinical classification. The mild phenotype is characterized by a delayed maturation from slow to fast muscle fibers. Other muscle transcripts, such as telethonin, myosin light-chains 3 and 1V, are underexpressed in this group. We suggest that expression profiling will provide important information to improve our understanding of the molecular basis of laminin alpha-2 positive MDC
Human MYO18B, a novel unconventional myosin heavy chain expressed in striated muscles moves into the myonuclei upon differentiation
No full textWe have characterized a novel unconventional myosin heavy chain, named MYO18B, that appears to be expressed mainly in human cardiac and skeletal muscles and, at lower levels, in testis. MYO18B transcript is detected in all types of striated muscles but at much lower levels compared to class II sarcomeric myosins, and it is up regulated after in vitro differentiation of myoblasts into myotubes. Phylogenetic analysis shows that this myosin belongs to the recently identified class XVIII, however, unlike the other member of this class, it seems to be unique to Vertebrate since it contains two large amino acid domains of unknown function at the N and C-termini. Immunolocalization of MYO18B protein in skeletal muscle cells shows that this myosin heavy chain is located in the cytoplasm of undifferentiated myoblasts. After in vitro differentiation into myotubes, a fraction of this protein is accumulated in a subset of myonuclei. This nuclear localization was confirmed by immunofluorescence experiments on primary cardiomyocytes and adult muscle sections. In the cytoplasm MYO18B shows a punctate staining, both in cardiac and skeletal fibers. In some cases, cardiomyocytes show a partial sarcomeric pattern of MYO18B alternating that of alpha-actinin-2. In skeletal muscle the cytoplasmic MYO18B results much more evident in the fast type fibers
- …
