Publications by authors named "Robson Francisco Carvalho"

56 Publications

Preventive training does not interfere with mRNA-encoding myosin and collagen expression during pulmonary arterial hypertension.

PLoS One 2021 8;16(9):e0244768. Epub 2021 Sep 8.

Postgraduate Program in Animal Science, University of Western São Paulo (UNOESTE), Presidente Prudente, São Paulo, Brazil.

To gain insight on the impact of preventive exercise during pulmonary arterial hypertension (PAH), we evaluated the gene expression of myosins and gene-encoding proteins associated with the extracellular matrix remodeling of right hypertrophied ventricles. We used 32 male Wistar rats, separated in four groups: Sedentary Control (S, n = 8); Control with Training (T, n = 8); Sedentary with Pulmonary Arterial Hypertension (SPAH, n = 8); and Pulmonary Arterial Hypertension with Training (TPAH, n = 8). All rats underwent a two-week adaptation period; T and TPAH group rats then proceeded to an eight-week training period on a treadmill. At the beginning of the 11th week, S and T groups received an intraperitoneal injection of saline, and SPAH and TPAH groups received an injection of monocrotaline (60 mg/kg). Rats in the T and TPAH groups then continued with the training protocol until the 13th week. We assessed exercise capacity, echocardiography analysis, Fulton's index, cross-sectional areas of cardiomyocytes, collagen content and types, and fractal dimension (FD). Transcript abundance of myosins and extracellular matrix genes were estimated through reverse transcription-quantitative PCR (RT-qPCR). When compared to the SPAH group, the TPAH group showed increases in functional capacity and pulmonary artery acceleration time/pulmonary ejection time ratio and decreases in Fulton's index and cross-sectional areas of myocyte cells. However, preventive exercise did not induce alterations in col1a1 and myh7 gene expression. Our findings demonstrate that preventive exercise improved functional capacity, reduced cardiac hypertrophy, and attenuated PH development without interfering in mRNA-encoding myosin and collagen expression during PAH.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244768PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425576PMC
September 2021

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth.

PLoS One 2021 22;16(7):e0255006. Epub 2021 Jul 22.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350-320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus-Ostariophysi) and Nile tilapias (Oreochromis niloticus-Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297816PMC
July 2021

TLR4 deficiency upregulates TLR9 expression and enhances irinotecan-related intestinal mucositis and late-onset diarrhoea.

Br J Pharmacol 2021 Jul 3. Epub 2021 Jul 3.

Laboratory of Inflammation and Cancer Pharmacology, Drug Research and Development Center (NPDM), Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará, Brazil.

Background And Purpose: Severe diarrhoea, a common gastrointestinal manifestation of anticancer treatment with irinotecan, might involve single nucleotide polymorphisms (SNPs) of toll-like receptors (TLRs), described as critical bacterial sensors in the gut. Here, colorectal cancer patients carrying missense TLR4 A896G (rs4986790) or C1,196T (rs4986791) SNPs and Tlr4 knockout (Tlr4-/-) mice were given irinotecan to investigate the severity of the induced diarrhoea.

Experimental Approach: Forty-six patients treated with irinotecan-based regimens had diarrhoea severity analysed according to TLR4 genotypes. In the experimental setting, wild-type (WT) or Tlr4-/- mice were given irinotecan (45 or 75 mg·kg , i.p.) or saline (3 ml·kg ). Diarrhoea severity was evaluated by measuring intestinal injury and inflammatory markers expression after animals were killed.

Key Results: All patients with TLR4 SNPs chemotherapy-treated presented diarrhoea, whereas gastrointestinal toxicity was observed in 50% of the wild homozygous individuals. Mice injected with irinotecan presented systemic bacterial translocation and increased TLR4 immunostaining in the intestine. In line with the clinical findings, Tlr4 gene deficiency enhanced irinotecan-related diarrhoea and TLR9 expression in mice. An increased myeloperoxidase activity and Il-18 expression along with IL-10 decreased production in Tlr4-/- mice also indicated an intensified intestinal damage and inflammatory response.

Conclusion And Implications: TLR4 deficiency upregulates TLR9 expression and enhances intestinal damage and the severity of late-onset diarrhoea during irinotecan-based treatment. Identifying patients genetically predisposed to chemotherapy-associated diarrhoea is a strategy toward precision medicine.
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http://dx.doi.org/10.1111/bph.15609DOI Listing
July 2021

Maternal protein restriction changes structural and metabolic gene expression in the skeletal muscle of aging offspring rats.

Histol Histopathol 2021 Apr 12:18337. Epub 2021 Apr 12.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Maternal protein restriction affects postnatal skeletal muscle physiology with impacts that last through senility. To investigate the morphological and molecular characteristics of skeletal muscle in aging rats subjected to maternal protein restriction, we used aged male rats (540 days old) born of dams fed a protein restricted diet (6% protein) during pregnancy and lactation. Using morphological, immunohistochemical and molecular analyses, we evaluated the soleus (SOL) and extensor digitorum longus (EDL) muscles, muscle fiber cross-sectional area (CSA) (n=8), muscle fiber frequency (n=5) and the gene expression (n=8) of the oxidative markers (succinate dehydrogenase-Sdha and citrate synthase-CS) and the glycolytic marker (lactate dehydrogenase-Ldha). Global transcriptome analysis (n=3) was also performed to identify differentially regulated genes, followed by gene expression validation (n=8). The oxidative SOL muscle displayed a decrease in muscle fiber CSA (*p<0.05) and in the expression of oxidative metabolism marker Sdha (***p<0.001), upregulation of the anabolic Igf-1 (**p<0.01), structural Chad (**p<0.01), and Fmod (*p<0.05) genes, and downregulation of the Hspb7 (**p<0.01) gene. The glycolytic EDL muscle exhibited decreased IIA (*p<0.05) and increased IIB (*p<0.05) fiber frequency, and no changes in muscle fiber CSA or in the expression of oxidative metabolism genes. In contrast, the gene expression of Chad (**p<0.01) was upregulated and the Myog (**p<0.01) gene was downregulated. Collectively, our morphological, immunohistochemical and molecular analyses showed that maternal protein restriction induced changes in the expression of metabolic, anabolic, myogenic, and structural genes, mainly in the oxidative SOL muscle, in aged offspring rats.
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http://dx.doi.org/10.14670/HH-18-337DOI Listing
April 2021

Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu () Juveniles: In Vivo and In Vitro Studies.

Int J Mol Sci 2021 Mar 15;22(6). Epub 2021 Mar 15.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, UNESP, Botucatu 18618-689, São Paulo, Brazil.

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu () following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.
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http://dx.doi.org/10.3390/ijms22062995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998472PMC
March 2021

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BMC Biol 2021 03 19;19(1):52. Epub 2021 Mar 19.

Departamento de Genética, Universidad de Granada, 18071, Granada, Spain.

Background: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested.

Results: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction.

Conclusions: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.
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http://dx.doi.org/10.1186/s12915-021-00991-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7976721PMC
March 2021

Prediction of SARS-CoV Interaction with Host Proteins during Lung Aging Reveals a Potential Role for TRIB3 in COVID-19.

Aging Dis 2021 Feb 1;12(1):42-49. Epub 2021 Feb 1.

1Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

COVID-19 is prevalent in the elderly. Old individuals are more likely to develop pneumonia and respiratory failure due to alveolar damage, suggesting that lung senescence may increase the susceptibility to SARS-CoV-2 infection and replication. Considering that human coronavirus (HCoVs; SARS-CoV-2 and SARS-CoV) require host cellular factors for infection and replication, we analyzed Genotype-Tissue Expression (GTEx) data to test whether lung aging is associated with transcriptional changes in human protein-coding genes that potentially interact with these viruses. We found decreased expression of the gene tribbles homolog 3 () during aging in male individuals, and its protein was predicted to interact with HCoVs nucleocapsid protein and RNA-dependent RNA polymerase. Using publicly available lung single-cell data, we found expressed mainly in alveolar epithelial cells that express SARS-CoV-2 receptor ACE2. Functional enrichment analysis of age-related genes, in common with SARS-CoV-induced perturbations, revealed genes associated with the mitotic cell cycle and surfactant metabolism. Given that TRIB3 was previously reported to decrease virus infection and replication, the decreased expression of in aged lungs may help explain why older male patients are related to more severe cases of the COVID-19. Thus, drugs that stimulate TRIB3 expression should be evaluated as a potential therapy for the disease.
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http://dx.doi.org/10.14336/AD.2020.1112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801268PMC
February 2021

Transcriptome of Two Canine Prostate Cancer Cells Treated With Toceranib Phosphate Reveals Distinct Antitumor Profiles Associated With the PDGFR Pathway.

Front Vet Sci 2020 26;7:561212. Epub 2020 Nov 26.

Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, Brazil.

Canine prostate cancer (PC) presents a poor antitumor response, usually late diagnosis and prognosis. Toceranib phosphate (TP) is a nonspecific inhibitor of receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and c-KIT. This study aimed to evaluate VEGFR2, PDGFR-β, and c-KIT protein expression in two established canine PC cell lines (PC1 and PC2) and the transcriptome profile of the cells after treatment with TP. Immunofluorescence (IF) analysis revealed VEGFR2 and PDGFR-β protein expression and the absence of c-KIT protein expression in both cell lines. After TP treatment, only the viability of PC1 cells decreased in a dose-dependent manner. Transcriptome and enrichment analyses of treated PC1 cells revealed 181 upregulated genes, which were related to decreased angiogenesis and cell proliferation. In addition, we found upregulated β, and expression in PC1 cells, and the upregulation of β was also observed in treated PC1 cells by qPCR. PC2 cells had fewer protein-protein interactions (PPIs), with 18 upregulated and 22 downregulated genes; the upregulated genes were involved in the regulation of parallel pathways and mechanisms related to proliferation, which could be associated with the resistance observed after treatment. The canine PC1 cell line but not the PC2 cell line showed decreased viability after treatment with TP, although both cell lines expressed PDGFR and VEGFR receptors. Further studies could explain the mechanism of resistance in PC2 cells and provide a basis for personalized treatment for dogs with PC.
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http://dx.doi.org/10.3389/fvets.2020.561212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726326PMC
November 2020

Identification of potential molecular pathways involved in prostate carcinogenesis in offspring exposed to maternal malnutrition.

Aging (Albany NY) 2020 10 13;12(20):19954-19978. Epub 2020 Oct 13.

Department of Structural and Functional Biology, Institute of Biosciences, Sao Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

The developmental origins of health and disease concept links adult diseases with early-life exposure to inappropriate environmental conditions. Intrauterine and postnatal malnutrition may lead to an increased incidence of type 2 diabetes, obesity, and cardiovascular diseases. Maternal malnutrition (MM) has also been associated with prostate carcinogenesis. However, the molecular mechanisms associated with this condition remain poorly understood. Using a proteomic analysis, we demonstrated that MM changed the levels of proteins associated with growth factors, estrogen signaling, detoxification, and energy metabolism in the prostate of both young and old rats. These animals also showed increased levels of molecular markers of endoplasmic reticulum function and histones. We further performed an analysis that identified commonly deregulated proteins in the ventral prostate of old rats submitted to MM with a mouse model and patients with prostate cancer. In conclusion, our results demonstrated that estrogenic signaling pathways, endoplasmic reticulum functions, energy metabolism, and molecular sensors of protein folding and Ca2+ homeostasis, besides histone, and RAS-GTPase family appear to be involved in this process. Knowledge of these factors may raise discussions regarding the role of maternal dietary intervention as a public policy for the lifelong prevention of chronic diseases.
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http://dx.doi.org/10.18632/aging.104093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655221PMC
October 2020

Comparison of microRNA Expression Profile in Chronic Myeloid Leukemia Patients Newly Diagnosed and Treated by Allogeneic Hematopoietic Stem Cell Transplantation.

Front Oncol 2020 4;10:1544. Epub 2020 Sep 4.

Department of Internal Medicine, São Paulo State University (UNESP-FMB), Botucatu, Brazil.

Chronic myeloid leukemia (CML) results from a translocation between chromosomes 9 and 22, which generates the Philadelphia chromosome. This forms BCR/ABL1, an active tyrosine kinase protein that promotes cell growth and replication. Despite great progress in CML treatment in the form of tyrosine kinase inhibitors, allogeneic-hematopoietic stem cell transplantation (allo-HSCT) is currently used as an important treatment alternative for patients resistant to these inhibitors. Studies have shown that unregulated expression of microRNAs, which act as oncogenes or tumor suppressors, is associated with human cancers. This contributes to tumor formation and development by stimulating proliferation, angiogenesis, and invasion. Research has demonstrated the potential of microRNAs as biomarkers for cancer diagnosis, prognosis, and therapeutic targets. In the present study, we compared the circulating microRNA expression profiles of 14 newly diagnosed patients with chronic phase-CML and 14 Philadelphia chromosome-negative patients after allo-HSCT. For each patient, we tested 758 microRNAs by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. The global expression profile of microRNAs revealed 16 upregulated and 30 downregulated microRNAs. Target genes were analyzed, and key pathways were extracted and compared. Bioinformatics tools were used to analyze data. Among the downregulated miRNA target genes, some genes related to cell proliferation pathways were identified. These results reveal the comprehensive microRNA profile of CML patients and the main pathways related to the target genes of these miRNAs in cytogenetic remission after allo-HSCT. These results provide new resources for exploring stem cell transplantation-based CML treatment strategies.
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http://dx.doi.org/10.3389/fonc.2020.01544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500210PMC
September 2020

The authors reply: Comment on "The expression landscape of cachexia-inducing factors in human cancers" by Freire et al.

J Cachexia Sarcopenia Muscle 2020 12 30;11(6):1854-1857. Epub 2020 Sep 30.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, UNESP, Botucatu, Brazil.

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http://dx.doi.org/10.1002/jcsm.12635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749551PMC
December 2020

A meta-analysis of microRNA networks regulated by melatonin in cancer: Portrait of potential candidates for breast cancer treatment.

J Pineal Res 2020 Nov 19;69(4):e12693. Epub 2020 Sep 19.

Department of Cell Systems and Anatomy, UT Health, San Antonio, TX, USA.

Melatonin is a ubiquitous molecule with a broad spectrum of functions including widespread anti-cancer activities. Identifying how melatonin intervenes in complex molecular signaling at the gene level is essential to guide proper therapies. Using meta-analysis approach, herein we examined the role of melatonin in regulating the expression of 46 microRNAs (miRNAs) and their target genes in breast, oral, gastric, colorectal, and prostate cancers, and glioblastoma. The deregulated miRNA-associated target genes revealed their involvement in the regulation of cellular proliferation, differentiation, apoptosis, senescence, and autophagy. Melatonin changes the expression of miRNA-associated genes in breast, gastric, and oral cancers. These genes are associated with cellular senescence, the hedgehog signaling pathway, cell proliferation, p53 signaling, and the hippo signaling pathway. Conversely, colorectal and prostate cancers as well as glioblastoma and oral carcinoma present a clear pattern of less pronounced changes in the expression of miRNA-associated genes. Most notably, colorectal cancer displayed a unique molecular change in response to melatonin. Considering breast cancer network complexity, we compared the genes found during the meta-analysis with RNA-Seq data from breast cancer-bearing mice treated with melatonin. Mechanistically, melatonin upregulated genes associated with immune responses and apoptotic processes, whereas it downregulated genes involved in cellular aggressiveness/metastasis (eg, mitosis, telomerase activity, and angiogenesis). We further characterized the expression profile of our gene subsets with human breast cancer and found eight upregulated genes and 16 downregulated genes that were appositively correlated with melatonin. Our results pose a multi-dimension network of tumor-associated genes regulated by miRNAs potentially targeted by melatonin.
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http://dx.doi.org/10.1111/jpi.12693DOI Listing
November 2020

Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response through a HIF-1α/Glycolysis-Dependent Axis.

Cell Metab 2020 09 17;32(3):437-446.e5. Epub 2020 Jul 17.

Department of Internal Medicine, School of Medical Sciences, University of Campinas, Campinas, São Paulo, Brazil.

COVID-19 can result in severe lung injury. It remained to be determined why diabetic individuals with uncontrolled glucose levels are more prone to develop the severe form of COVID-19. The molecular mechanism underlying SARS-CoV-2 infection and what determines the onset of the cytokine storm found in severe COVID-19 patients are unknown. Monocytes and macrophages are the most enriched immune cell types in the lungs of COVID-19 patients and appear to have a central role in the pathogenicity of the disease. These cells adapt their metabolism upon infection and become highly glycolytic, which facilitates SARS-CoV-2 replication. The infection triggers mitochondrial ROS production, which induces stabilization of hypoxia-inducible factor-1α (HIF-1α) and consequently promotes glycolysis. HIF-1α-induced changes in monocyte metabolism by SARS-CoV-2 infection directly inhibit T cell response and reduce epithelial cell survival. Targeting HIF-1ɑ may have great therapeutic potential for the development of novel drugs to treat COVID-19.
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http://dx.doi.org/10.1016/j.cmet.2020.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367032PMC
September 2020

MicroRNA-mRNA Co-sequencing Identifies Transcriptional and Post-transcriptional Regulatory Networks Underlying Muscle Wasting in Cancer Cachexia.

Front Genet 2020 29;11:541. Epub 2020 May 29.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, Botucatu, Brazil.

Cancer cachexia is a metabolic syndrome with alterations in gene regulatory networks that consequently lead to skeletal muscle wasting. Integrating microRNAs-mRNAs omics profiles offers an opportunity to understand transcriptional and post-transcriptional regulatory networks underlying muscle wasting. Here, we used RNA sequencing to simultaneously integrate and explore microRNAs and mRNAs expression profiles in the tibialis anterior (TA) muscles of the Lewis Lung Carcinoma (LLC) model of cancer cachexia. We found 1,008 mRNAs and 18 microRNAs differentially expressed in cachectic mice compared with controls. Although our transcriptomic analysis demonstrated a high heterogeneity in mRNA profiles of cachectic mice, we identified a reduced number of differentially expressed genes that were uniformly regulated within cachectic muscles. This set of uniformly regulated genes is associated with the extracellular matrix (ECM), proteolysis, and inflammatory response. We also used transcriptomic data to perform enrichment analysis of transcriptional factor binding sites in promoter sequences, which revealed activation of the atrophy-related transcription factors NF-κB, Stat3, AP-1, and FoxO. Furthermore, the integration of mRNA and microRNA expression profiles identified post-transcriptional regulation by microRNAs of genes involved in ECM organization, cell migration, transcription factors binding, ion transport, and the FoxO signaling pathway. Our integrative analysis of microRNA-mRNA co-profiles comprehensively characterized regulatory relationships of molecular pathways and revealed microRNAs targeting ECM-associated genes in cancer cachexia.
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http://dx.doi.org/10.3389/fgene.2020.00541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272700PMC
May 2020

An Integrated Meta-Analysis of Secretome and Proteome Identify Potential Biomarkers of Pancreatic Ductal Adenocarcinoma.

Cancers (Basel) 2020 03 18;12(3). Epub 2020 Mar 18.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

Pancreatic ductal adenocarcinoma (PDAC) is extremely aggressive, has an unfavorable prognosis, and there are no biomarkers for early detection of the disease or identification of individuals at high risk for morbidity or mortality. The cellular and molecular complexity of PDAC leads to inconsistences in clinical validations of many proteins that have been evaluated as prognostic biomarkers of the disease. The tumor secretome, a potential source of biomarkers in PDAC, plays a crucial role in cell proliferation and metastasis, as well as in resistance to treatments, which together contribute to a worse clinical outcome. The massive amount of proteomic data from pancreatic cancer that has been generated from previous studies can be integrated and explored to uncover secreted proteins relevant to the diagnosis and prognosis of the disease. The present study aimed to perform an integrated meta-analysis of PDAC proteome and secretome public data to identify potential biomarkers of the disease. Our meta-analysis combined mass spectrometry data obtained from two systematic reviews of the pancreatic cancer literature, which independently selected 20 studies of the secretome and 35 of the proteome. Next, we predicted the secreted proteins using seven in silico tools or databases, which identified 39 secreted proteins shared between the secretome and proteome data. Notably, the expression of 31 genes of these secretome-related proteins was upregulated in PDAC samples from The Cancer Genome Atlas (TCGA) when compared to control samples from TCGA and The Genotype-Tissue Expression (GTEx). The prognostic value of these 39 secreted proteins in predicting survival outcome was confirmed using gene expression data from four PDAC datasets (validation set). The gene expression of these secreted proteins was able to distinguish high- and low-survival patients in nine additional tumor types from TCGA, demonstrating that deregulation of these secreted proteins may also contribute to the prognosis in multiple cancers types. Finally, we compared the prognostic value of the identified secreted proteins in PDAC biomarkers studies from the literature. This analysis revealed that our gene signature performed equally well or better than the signatures from these previous studies. In conclusion, our integrated meta-analysis of PDAC proteome and secretome identified 39 secreted proteins as potential biomarkers, and the tumor gene expression profile of these proteins in patients with PDAC is associated with worse overall survival.
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http://dx.doi.org/10.3390/cancers12030716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140071PMC
March 2020

The expression landscape of cachexia-inducing factors in human cancers.

J Cachexia Sarcopenia Muscle 2020 08 3;11(4):947-961. Epub 2020 Mar 3.

Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University, UNESP, Botucatu, Brazil.

Background: Cachexia is a multifactorial syndrome highly associated with specific tumour types, but the causes of variation in cachexia prevalence and severity are unknown. While circulating plasma mediators (soluble cachectic factors) derived from tumours have been implicated with the pathogenesis of the syndrome, these associations were generally based on plasma concentration rather than tissue-specific gene expression levels. Here, we hypothesized that tumour gene expression profiling of cachexia-inducing factors (CIFs) in human cancers with different prevalence of cachexia could reveal potential cancer-specific cachexia mediators and biomarkers of clinical outcome.

Methods: First, we combined uniformly processed RNA sequencing data from The Cancer Genome Atlas and Genotype-Tissue Expression databases to characterize the expression profile of secretome genes in 12 cancer types (4651 samples) compared with their matched normal tissues (2737 samples). We systematically investigated the transcriptomic data to assess the tumour expression profile of 25 known CIFs and their predictive values for patient survival. We used the Xena Functional Genomics tool to analyse the gene expression of CIFs according to neoplastic cellularity in pancreatic adenocarcinoma, which is known to present the highest prevalence of cachexia.

Results: A comprehensive characterization of the expression profiling of secreted genes in different human cancers revealed pathways and mediators with a potential role in cachexia within the tumour microenvironment. Cytokine-related and chemokine-related pathways were enriched in tumour types frequently associated with the syndrome. CIFs presented a tumour-specific expression profile, in which the number of upregulated genes was correlated with the cachexia prevalence (r : 0.80; P value: 0.002) and weight loss (r : 0.81; P value: 0.002). The distinct gene expression profile, according to tumour type, was significantly associated with prognosis (P value ≤ 1.96 E-06). In pancreatic adenocarcinoma, the upregulated CIF genes were associated with tumours presenting low neoplastic cellularity and high leucocyte fraction and not with tumour grade.

Conclusions: Our results present a biological dimension of tumour-secreted elements that are potentially useful to explain why specific cancer types are more likely to develop cachexia. The tumour-specific profile of CIFs may help the future development of better targeted therapies to treat cancer types highly associated with the syndrome.
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http://dx.doi.org/10.1002/jcsm.12565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432594PMC
August 2020

Increased DSG2 plasmatic levels identified by transcriptomic-based secretome analysis is a potential prognostic biomarker in laryngeal carcinoma.

Oral Oncol 2020 04 19;103:104592. Epub 2020 Feb 19.

Department of Clinical Genetics, University Hospital, Institute of Regional Health Research, University of Southern Denmark, Vejle, Denmark. Electronic address:

Objectives: The tumor secretome deconvolution is a promising strategy to identify diagnostic and prognostic biomarkers. Here, transcriptomic-based secretome analysis was performed aiming to discover laryngeal squamous cell carcinomas (LSCC) biomarkers from potentially secreted proteins (PSPs).

Material And Methods: The tumor expression profile (35 LSCC biopsies compared with surrounding normal tissues - SN) revealed 589 overexpressed genes. This gene list was used for secretome analysis based on laryngeal tumors and related secretome databases.

Results: Forty-nine (Laryngeal tumor secretome database) and 50 (Human Protein Atlas and Cancer Secretome Database) PSPs presented an association with worse overall survival. Specifically, DSG2 overexpression was strongly correlated with poor survival and distant metastasis. DSG2 increased expression was confirmed in the LSCC dataset (LSCC = 111; SN = 12) from TCGA. A significant association between shorter survival and DSG2 overexpression was also detected. In an independent cohort of cases, we analyzed and confirmed high protein levels of DSG2 in plasma from LSCC patients.

Conclusion: A set of PSPs including the circulating DSG2, were associated with shorter overall survival in LSCC. DSG2 overexpression was also correlated with distant metastasis. The high plasmatic protein levels of DSG2 suggest its potential to be tested in liquid biopsies and applied as prognostic biomarker of LSCC.
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http://dx.doi.org/10.1016/j.oraloncology.2020.104592DOI Listing
April 2020

Prolonged fasting followed by refeeding modifies proteome profile and parvalbumin expression in the fast-twitch muscle of pacu (Piaractus mesopotamicus).

PLoS One 2019 19;14(12):e0225864. Epub 2019 Dec 19.

Department of Morphology, Institute of Bioscience of Botucatu, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Here, we analyzed the fast-twitch muscle of juvenile Piaractus mesopotamicus (pacu) submitted to prolonged fasting (30d) and refeeding (6h, 24h, 48h and 30d). We measured the relative rate of weight and length increase (RRIlength and RRIweight), performed shotgun proteomic analysis and did Western blotting for PVALB after 30d of fasting and 30d of refeeding. We assessed the gene expression of igf-1, mafbx and pvalb after 30d of fasting and after 6h, 24h, 48h and 30d of refeeding. We performed a bioinformatic analysis to predict miRNAs that possibly control parvalbumin expression. After fasting, RRIlength, RRIweight and igf-1 expression decreased, while the mafbx expression increased, which suggest that prolonged fasting caused muscle atrophy. After 6h and 24h of refeeding, mafbx was not changed and igf-1 was downregulated, while after 48h of refeeding mafbx was downregulated and igf-1 was not changed. After 30d of refeeding, RRIlength and RRIweight were increased and igf-1 and mafbx expression were not changed. Proteomic analysis identified 99 proteins after 30d of fasting and 71 proteins after 30d of refeeding, of which 23 and 17, respectively, were differentially expressed. Most of these differentially expressed proteins were related to cytoskeleton, muscle contraction, and metabolism. Among these, parvalbumin (PVALB) was selected for further validation. The analysis showed that pvalb mRNA was downregulated after 6h and 24h of refeeding, but was not changed after 30d of fasting or 48h and 30d of refeeding. The Western blotting confirmed that PVALB protein was downregulated after 30d of fasting and 30d of refeeding. The downregulation of the protein and the unchanged expression of the mRNA after 30d of fasting and 30d of refeeding suggest a post-transcriptional regulation of PVALB. Our miRNA analysis predicted 444 unique miRNAs that may target pvalb. In conclusion, muscle atrophy and partial compensatory growth caused by prolonged fasting followed by refeeding affected the muscle proteome and PVALB expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225864PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922423PMC
March 2020

Tumor Transcriptome Reveals High Expression of IL-8 in Non-Small Cell Lung Cancer Patients with Low Pectoralis Muscle Area and Reduced Survival.

Cancers (Basel) 2019 08 26;11(9). Epub 2019 Aug 26.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 18618-689, São Paulo, Brazil.

Cachexia is a syndrome characterized by an ongoing loss of skeletal muscle mass associated with poor patient prognosis in non-small cell lung cancer (NSCLC). However, prognostic cachexia biomarkers in NSCLC are unknown. Here, we analyzed computed tomography (CT) images and tumor transcriptome data to identify potentially secreted cachexia biomarkers (PSCB) in NSCLC patients with low-muscularity. We integrated radiomics features (pectoralis muscle, sternum, and tenth thoracic (T10) vertebra) from CT of 89 NSCLC patients, which allowed us to identify an index for screening muscularity. Next, a tumor transcriptomic-based secretome analysis from these patients (discovery set) was evaluated to identify potential cachexia biomarkers in patients with low-muscularity. The prognostic value of these biomarkers for predicting recurrence and survival outcome was confirmed using expression data from eight lung cancer datasets (validation set). Finally, C2C12 myoblasts differentiated into myotubes were used to evaluate the ability of the selected biomarker, interleukin (IL)-8, in inducing muscle cell atrophy. We identified 75 over-expressed transcripts in patients with low-muscularity, which included and . Also, we identified , , , , , , and as PSCB in the tumor secretome. These PSCB were capable of distinguishing worse and better prognosis (recurrence and survival) in NSCLC patients. was confirmed as a predictor of worse prognosis in all validation sets. In vitro assays revealed that IL-8 promoted C2C12 myotube atrophy. Tumors from low-muscularity patients presented a set of upregulated genes encoding for secreted proteins, including pro-inflammatory cytokines that predict worse overall survival in NSCLC. Among these upregulated genes, expression in NSCLC tissues was associated with worse prognosis, and the recombinant IL-8 was capable of triggering atrophy in C2C12 myotubes.
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http://dx.doi.org/10.3390/cancers11091251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769884PMC
August 2019

Locally advanced rectal cancer transcriptomic-based secretome analysis reveals novel biomarkers useful to identify patients according to neoadjuvant chemoradiotherapy response.

Sci Rep 2019 06 18;9(1):8702. Epub 2019 Jun 18.

Department of Clinical Genetics, University Hospital of Southern Denmark, Vejle, 7100, Denmark.

Most patients with locally advanced rectal cancer (LARC) present incomplete pathological response (pIR) to neoadjuvant chemoradiotherapy (nCRT). Despite the efforts to predict treatment response using tumor-molecular features, as differentially expressed genes, no molecule has proved to be a strong biomarker. The tumor secretome analysis is a promising strategy for biomarkers identification, which can be assessed using transcriptomic data. We performed transcriptomic-based secretome analysis to select potentially secreted proteins using an in silico approach. The tumor expression profile of 28 LARC biopsies collected before nCRT was compared with normal rectal tissues (NT). The expression profile showed no significant differences between complete (pCR) and incomplete responders to nCRT. Genes with increased expression (pCR = 106 and pIR = 357) were used for secretome analysis based on public databases (Vesiclepedia, Human Cancer Secretome, and Plasma Proteome). Seventeen potentially secreted candidates (pCR = 1, pIR = 13 and 3 in both groups) were further investigated in two independent datasets (TCGA and GSE68204) confirming their over-expression in LARC and association with nCRT response (GSE68204). The expression of circulating amphiregulin and cMET proteins was confirmed in serum from 14 LARC patients. Future studies in liquid biopsies could confirm the utility of these proteins for personalized treatment in LARC patients.
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http://dx.doi.org/10.1038/s41598-019-45151-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582145PMC
June 2019

The combination of resveratrol and exercise enhances muscle growth characteristics in pacu (Piaractus mesopotamicus).

Comp Biochem Physiol A Mol Integr Physiol 2019 09 8;235:46-55. Epub 2019 May 8.

Department of Morphology, Institute of Bioscience, Sao Paulo State University, UNESP, Botucatu, SP, Brazil; Aquaculture Center, CAUNESP, Sao Paulo State University, UNESP, Jaboticabal, SP, Brazil. Electronic address:

Pacu is a tropical fish with important value to aquaculture. During cellular metabolism, reactive oxygen species (ROS) are produced, which can influence muscle growth. Resveratrol is an effective antioxidant that scavenges ROS and can modulate physical performance preventing oxidative stress. We investigated the effects of resveratrol and exercise on pacu muscle growth characteristics. Four groups were used: fish fed with control diet /without exercise (C); fish fed with control diet/subjected to exercise (CE); fish fed resveratrol-supplemented diet/without exercise (R); and fish fed resveratrol-supplemented diet/subjected to exercise (RE). At 30 days, the RE group presented a significant increase in body weight, fewer muscle fibers in the 20-40 μm and more fibers in the >60 μm diameter class compared to the C group. At day 7, catalase activity decreased in CE and RE groups. Superoxide dismutase activity decreased only in the CE group. Myod and mtor gene expression was higher in R and RE and igf-1 was up-regulated in the RE group. Murf1a level decreased in CE, R, and RE, while sdha expression was higher in the RE group. We suggest that resveratrol in combination with exercise was beneficial for muscle growth and metabolism, increasing the expression levels of genes related to muscle anabolism and oxidative metabolism, besides the decrease of catabolic gene expression. Notably, all of these changes occurred together with muscle hypertrophy and increased body weight. Our results show a positive application for resveratrol in association with exercise as a strategy to improve the growth performance of juvenile pacus.
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http://dx.doi.org/10.1016/j.cbpa.2019.05.002DOI Listing
September 2019

Proteomic analysis of the fast-twitch muscle of pacu (Piaractus mesopotamicus) after prolonged fasting and compensatory growth.

Comp Biochem Physiol Part D Genomics Proteomics 2019 06 24;30:321-332. Epub 2019 Apr 24.

Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Botucatu, Brazil. Electronic address:

Protocols that improve growth performance in fish while assuring product quality are important for aquaculture. Fasting followed by refeeding may promote compensatory growth, thus optimizing growth performance. During fasting and refeeding, fast-twitch muscle, which comprises most of fish fillet, undergoes intense plasticity. In this work, we studied the proteome of pacu (Piaractus mesopotamicus) fast-twitch muscle after 30 days of fasting (D30), 30 days of refeeding (D60) and 60 days of refeeding (D90) with two-dimensional electrophoresis, mass spectrometry and bioinformatics. Body mass, growth rate and muscle histology were also assessed. At D30, fish presented muscle catabolism and decreased growth. Proteomic analysis showed that metabolism proteins were the most affected, up and downregulated. Cytoskeleton and amino acid biosynthesis proteins were downregulated, while nuclear and regulatory proteins were upregulated. At D60, fish showed accelerated growth, despite the body mass not completely recovering. Metabolism proteins were still the most affected. Amino acid biosynthesis proteins became upregulated, while cytoskeleton proteins remained downregulated. At D90, the fish presented total compensatory growth. Many metabolic proteins were up or downregulated. Few cytoskeleton proteins remained differentially expressed. Amino acid biosynthesis proteins were mostly upregulated, but less than at D60. Prolonged fasting followed by refeeding also led to the regulation of possible meat quality biomarkers, such as antioxidant enzymes. This fact suggests possible consequences of this protocol on fish meat quality. Our work also enriches our knowledge on proteomic changes during muscle plasticity that occur during fasting and refeeding diet protocols.
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http://dx.doi.org/10.1016/j.cbd.2019.04.005DOI Listing
June 2019

The Pathway to Cancer Cachexia: MicroRNA-Regulated Networks in Muscle Wasting Based on Integrative Meta-Analysis.

Int J Mol Sci 2019 Apr 22;20(8). Epub 2019 Apr 22.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo 18.618-619, Brazil.

Cancer cachexia is a multifactorial syndrome that leads to significant weight loss. Cachexia affects 50%-80% of cancer patients, depending on the tumor type, and is associated with 20%-40% of cancer patient deaths. Besides the efforts to identify molecular mechanisms of skeletal muscle atrophy-a key feature in cancer cachexia-no effective therapy for the syndrome is currently available. MicroRNAs are regulators of gene expression, with therapeutic potential in several muscle wasting disorders. We performed a meta-analysis of previously published gene expression data to reveal new potential microRNA-mRNA networks associated with muscle atrophy in cancer cachexia. We retrieved 52 differentially expressed genes in nine studies of muscle tissue from patients and rodent models of cancer cachexia. Next, we predicted microRNAs targeting these differentially expressed genes. We also include global microRNA expression data surveyed in atrophying skeletal muscles from previous studies as background information. We identified deregulated genes involved in the regulation of apoptosis, muscle hypertrophy, catabolism, and acute phase response. We further predicted new microRNA-mRNA interactions, such as miR-27a/, miR-27a/, miR-27b/, miR-27b/, miR-140/, miR-199a/, and miR-199a/, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting , , and , which may be considered for the development of novel therapeutic strategies for cancer cachexia. Our study has broadened the knowledge of microRNA-regulated networks that are likely associated with muscle atrophy in cancer cachexia, pointing to their involvement as potential targets for novel therapeutic strategies.
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http://dx.doi.org/10.3390/ijms20081962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515458PMC
April 2019

Ascorbic acid stimulates the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus).

Sci Rep 2019 02 18;9(1):2229. Epub 2019 Feb 18.

São Paulo State University (UNESP), Institute of Biosciences, Department of Morphology, Botucatu, São Paulo, Brazil.

The postembryonic growth of skeletal muscle in teleost fish involves myoblast proliferation, migration and differentiation, encompassing the main events of embryonic myogenesis. Ascorbic acid plays important cellular and biochemical roles as an antioxidant and contributes to the proper collagen biosynthesis necessary for the structure of connective and bone tissues. However, whether ascorbic acid can directly influence the mechanisms of fish myogenesis and skeletal muscle growth remains unclear. The aim of our work was to evaluate the effects of ascorbic acid supplementation on the in vitro myoblast proliferation and migration of pacu (Piaractus mesopotamicus). To provide insight into the potential antioxidant role of ascorbic acid, we also treated myoblasts in vitro with menadione, which is a powerful oxidant. Our results show that ascorbic acid-supplemented myoblasts exhibit increased proliferation and migration and are protected against the oxidative stress caused by menadione. In addition, ascorbic acid increased the activity of the antioxidant enzyme superoxide dismutase and the expression of myog and mtor, which are molecular markers related to skeletal muscle myogenesis and protein synthesis, respectively. This work reveals a direct influence of ascorbic acid on the mechanisms of pacu myogenesis and highlights the potential use of ascorbic acid for stimulating fish skeletal muscle growth.
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http://dx.doi.org/10.1038/s41598-019-38536-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379551PMC
February 2019

Can the antral follicular count modulate the gene expression of bovine oviducts in Aberdeen Angus and Nelore heifers?

PLoS One 2018 29;13(8):e0202017. Epub 2018 Aug 29.

Departament of Animal Science, University of Western São Paulo (UNOESTE), Presidente Prudente, São Paulo, Brazil.

The number of visible ovarian antral follicles (antral follicle count-AFC) is repeatable in bovine individuals, but highly variable between animals, and with differences between Bos taurus and Bos indicus breeds. Several studies have tried to determine the correlation between AFC and increased fertility in cattle. While the impacts of AFC on embryo production, hormonal levels, and pregnancy rates have been described, the molecular effects of AFC on bovine oviducts have not yet been investigated. Here, the aim was to investigate the impact of breeds, such as Aberdeen Angus and Nelore heifer with high or low AFC, on abundance of transcripts and protein related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction in the oviducts. In summary, the ovulation side was the major factor that affected transcript abundance on bovine oviducts. However, a discreet effect among AFC and cattle breeds was also observed. Based on this, we concluded and reinforced here that differential microenvironments between ipsilateral and contralateral oviducts have a major effect on modulating the transcripts related to oviductal transport, sperm reservoir formation, monospermy control, and gamete interaction. However, we cannot exclude that there is minimal effect of AFC or breed on regulation of some genes (such as AGTR1, ACE1, FUCA1, and VEGFA) in bovine oviducts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202017PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114296PMC
January 2019

Recovery of Cardiac Remodeling and Dysmetabolism by Pancreatic Islet Injury Improvement in Diabetic Rats after Yacon Leaf Extract Treatment.

Oxid Med Cell Longev 2018 10;2018:1821359. Epub 2018 Apr 10.

Medical School, São Paulo State University (UNESP), Botucatu, SP, Brazil.

Yacon () is a native Andean plant rich in phenolic compounds, and its effects on dysmetabolism and cardiomyopathy in diabetic rats was evaluated. The rats (10/group) were allocated as follows: C, controls; C + Y, controls treated with Yacon leaf extract (YLE); DM, diabetic controls; and DM + Y, diabetic rats treated with YLE. Type 1 diabetes (T1DM) was induced by the administration of streptozotocin (STZ; 40 mg/kg body weight, single dose, i.p.), and treated groups received 100 mg/kg body weight YLE daily via gavage for 30 d. The YLE group shows an improvement in dysmetabolism and cardiomyopathy in the diabetic condition (DM versus DM + Y) promoting a significant reduction of glycemia by 63.39%, an increase in insulin concentration by 49.30%, and a decrease in serum triacylglycerol and fatty acid contents by 0.39- and 0.43-fold, respectively, by ameliorating the pancreatic islet injury, as well as increasing the activity of the antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) and decreasing the fibrosis and cellular disorganization in cardiac tissue. The apparent benefits of YLE seem to be mediated by ameliorating dysmetabolism and oxidative stress in pancreatic and cardiac tissues.
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http://dx.doi.org/10.1155/2018/1821359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051012PMC
October 2018

Osteoglycin post-transcriptional regulation by miR-155 induces cellular architecture changes in H9c2 cardiomyoblasts.

Gene 2018 Nov 7;676:9-15. Epub 2018 Jul 7.

Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil. Electronic address:

Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 significantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However, most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded. This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell morphology in physiological and pathological conditions.
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http://dx.doi.org/10.1016/j.gene.2018.07.020DOI Listing
November 2018

Fractal dimension analysis reveals skeletal muscle disorganization in mdx mice.

Biochem Biophys Res Commun 2018 09 6;503(1):109-115. Epub 2018 Jun 6.

Department of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil. Electronic address:

Duchenne Muscular Dystrophy (DMD) is characterized by muscle extracellular matrix disorganization due to the increased collagen deposition leading to fibrosis that significantly exacerbates disease progression. Fractal dimension analysis is a method that quantifies tissue/cellular disorganization and characterizes complex structures. The first objective of the present study was use fractal analysis to evaluate extracellular matrix disorganization in mdx mice soleus muscle. Next, we mimic a hyper-proliferation of fibrogenic cells by co-culturing NIH3T3 fibroblasts and C2C12 myoblasts to test whether fibroblasts induce disorganization in myoblast arrangement. Here, we show mdx presented high skeletal muscle disorganization as revealed by fractal analysis. Similarly, this method revealed that myoblasts co-cultured with fibroblast also presented cellular arrangement disorganization. We also reanalyzed skeletal muscle microarrays transcriptomic data from mdx and DMD patients that revealed transcripts related to extracellular matrix organization. This analysis also identified Osteoglycin, which was validated as a potential regulator of ECM organization in mdx dystrophic muscles. Our results demonstrate that fractal dimension is useful tool for the analysis of skeletal muscle disorganization in DMD and also reveal a fibroblast-myoblast cross-talk that contributes to "in vitro" myoblast disarrangement.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.189DOI Listing
September 2018

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

PLoS One 2017 21;12(11):e0188464. Epub 2017 Nov 21.

Department of Morphology, Institute of Biosciences, São Paulo State University, Botucatu, São Paulo, Brazil.

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80-90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3'UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3'UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188464PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697837PMC
December 2017
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