The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

RNA-seq analysis reveals modulation of inflammatory pathways by an enriched-triterpene natural extract in mouse and human macrophage cell lines.

Chronic inflammation is crucial in developing insulin resistance and type 2 diabetes. Previous studies have shown that a leaf extract of Eucalyptus tereticornis, with ursolic acid (UA), oleanolic acid (OA), and ursolic acid lactone (UAL) as the main molecules (78 %) mixed with unknown minor metabolites (22 %), provided superior anti-inflammatory, hypoglycemic, and hypolipidemic effects than reconstituted triterpenoid mixtures in macrophage cell lines and a pre-diabetic mouse model. Further identification of the molecular mechanisms of action of this mixture of triterpenes is required. This study aims to analyse the RNA expression profiles of mouse and human macrophage cell lines treated with the natural extract and its components. Activated macrophage cell lines were treated with the natural extract, UA, OA, UAL or a triterpene mixture (M1). RNA was extracted and sequenced using the DNBseq platform and the EnrichR software to perform gene enrichment analysis using the Gene Ontology database, Kyoto Encyclopedia of Genes and Genomes, and Reactome. To conduct clustering analysis, we standardised the normalised counts of each gene and applied k-means clustering. The combination of molecules in the natural extract has an additive or synergic effect that affects the expression of up-regulated genes by macrophage activation. Triterpenes (M1) regulated 76 % of human and 68 % of mouse genes, while uncharacterised minority molecules could regulate 24 % of human and 32 % of mouse genes. The extract inhibited the expression of many cytokines (IL6, IL1, OSM), chemokines (CXCL3), inflammatory mediators (MMP8 and MMP13) and the JAK-STAT signalling pathway in both models. The natural extract has a more powerful immunomodulatory effect than the triterpene mixture, increasing the number of genes regulated in mouse and human models. Our study shows that Eucalyptus tereticornis extract is a promising option for breaking the link between inflammation and insulin resistance.

Multi-omics characterization of the microbial populations and chemical space composition of a water kefir fermentation.

In recent years, the popularity of fermented foods has strongly increased based on their proven health benefits and the adoption of new trends among consumers. One of these health-promoting products is water kefir, which is a fermented sugary beverage based on kefir grains (symbiotic colonies of yeast, lactic acid and acetic acid bacteria). According to previous knowledge and the uniqueness of each water kefir fermentation, the following project aimed to explore the microbial and chemical composition of a water kefir fermentation and its microbial consortium, through the integration of culture-dependent methods, compositional metagenomics, and untargeted metabolomics. These methods were applied in two types of samples: fermentation grains (inoculum) and fermentation samples collected at different time points. A strains culture collection of ∼90 strains was established by means of culture-dependent methods, mainly consisting of individuals of Pichia membranifaciens, Acetobacter orientalis, Lentilactobacillus hilgardii, Lacticaseibacillus paracasei, Acetobacter pomorum, Lentilactobacillus buchneri, Pichia kudriavzevii, Acetobacter pasteurianus, Schleiferilactobacillus harbinensis, and Kazachstania exigua, which can be further studied for their use in synthetic consortia formulation. In addition, metabarcoding of each fermentation time was done by 16S and ITS sequencing for bacteria and yeast, respectively. The results show strong population shifts of the microbial community during the fermentation time course, with an enrichment of microbial groups after 72 h of fermentation. Metataxonomics results revealed Lactobacillus and Acetobacter as the dominant genera for lactic acid and acetic acid bacteria, whereas, for yeast, P. membranifaciens was the dominant species. In addition, correlation and systematic analyses of microbial growth patterns and metabolite richness allowed the recognition of metabolic enrichment points between 72 and 96 h and correlation between microbial groups and metabolite abundance (e.g., Bile acid conjugates and Acetobacter tropicalis). Metabolomic analysis also evidenced the production of bioactive compounds in this fermented matrix, which have been associated with biological activities, including antimicrobial and antioxidant. Interestingly, the chemical family of Isoschaftosides (C-glycosyl flavonoids) was also found, representing an important finding since this compound, with hepatoprotective and anti-inflammatory activity, had not been previously reported in this matrix. We conclude that the integration of microbial biodiversity, cultured species, and chemical data enables the identification of relevant microbial population patterns and the detection of specific points of enrichment during the fermentation process of a food matrix, which enables the future design of synthetic microbial consortia, which can be used as targeted probiotics for digestive and metabolic health.

Particulate matter impairs immune system function by up-regulating inflammatory pathways and decreasing pathogen response gene expression.

Airborne particulate matter produced by industrial sources and automobiles has been linked to increased susceptibility to infectious diseases and it is known to be recognized by cells of the immune system. The molecular mechanisms and changes in gene expression profiles induced in immune cells by PM have not been fully mapped out or systematically integrated. Here, we use RNA-seq to analyze mRNA profiles of human peripheral blood mononuclear cells after exposure to coarse particulate matter (PM10). Our analyses showed that PM10 was able to reprogram the expression of 1,196 genes in immune cells, including activation of a proinflammatory state with an increase in cytokines and chemokines. Activation of the IL-36 signaling pathway and upregulation of chemokines involved in neutrophil and monocyte recruitment suggest mechanisms for inflammation upon PM exposure, while NK cell-recruiting chemokines are repressed. PM exposure also increases transcription factors associated with inflammatory pathways (e.g., JUN, RELB, NFKB2, etc.) and reduces expression of RNases and pathogen response genes CAMP, DEFAs, AZU1, APOBEC3A and LYZ. Our analysis across gene regulatory and signaling pathways suggests that PM plays a role in the dysregulation of immune cell functions, relevant for antiviral responses and general host defense against pathogens.

Vitamin D modulates expression of antimicrobial peptides and proinflammatory cytokines to restrict Zika virus infection in macrophages.

Although the impact of Zika virus (ZIKV) infection on human health has been well documented, we still have no vaccine or effective treatment. This fact highlights the importance of searching for alternative therapy for treating ZIKV. To search for ZIKV antivirals, we examined the effect of vitamin D in monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D (D3-MDM) and explored the molecular mechanisms by analyzing transcriptional profiles. Our data show the restriction of ZIKV infection in D3-MDMs as compared to MDMs. Transcriptional profiles show that vitamin D alters about 19% of Zika response genes (8.2% diminished and 10.8% potentiated). Among the genes with diminished expression levels, we found proinflammatory cytokines and chemokines such as IL6, TNF, IL1A, IL1B, and IL12B, CCL1, CCL4, CCL7, CXCL3, CXCL6, and CXCL8. On the other hand, genes with potentiated expression were related to degranulation such as Lysozyme, cathelicidin (CAMP), and Serglycin. Since the CAMP gene encodes the antimicrobial peptide LL-37, we treated MDMs with LL-37 and infected them with ZIKV. The results showed a decrease in the proportion of infected cells. Our data provide new insights into the role of vitamin D in restricting ZIKV infection in macrophages that are mediated by induction of cathelicidin/LL-37 expression and downregulation of proinflammatory genes. Results highlight the biological relevance of vitamin D-inducible peptides as an antiviral treatment for Zika fever.

The Transcriptomic Landscape of Age-Induced Changes in Human Visceral Fat and the Predicted Omentum-Liver Connectome in Males.

Aging causes alterations in body composition. Specifically, visceral fat mass increases with age and is associated with age-related diseases. The pathogenic potential of visceral fat accumulation has been associated with its anatomical location and metabolic activity. Visceral fat may control systemic metabolism by secreting molecules that act in distal tissues, mainly the liver, through the portal vein. Currently, little is known about age-related changes in visceral fat in humans. Aiming to identify molecular and cellular changes occurring with aging in the visceral fat of humans, we analyzed publicly available transcriptomic data of 355 omentum samples from the Genotype-Tissue Expression portal (GTEx) of 20-79-year-old males and females. We identified the functional enrichment of genes associated with aging, inferred age-related changes in visceral fat cellularity by deconvolution analysis, profiled the senescence-associated secretory phenotype of visceral adipose tissue, and predicted the connectivity of the age-induced visceral fat secretome with the liver. We demonstrate that age induces alterations in visceral fat cellularity, synchronous to changes in metabolic pathways and a shift toward a pro-inflammatory secretory phenotype. Furthermore, our approach identified candidates such as ADIPOQ-ADIPOR1/ADIPOR2, FCN2-LPR1, and TF-TFR2 to mediate visceral fat-liver crosstalk in the context of aging. These findings cast light on how alterations in visceral fat with aging contribute to liver dysfunction and age-related disease etiology.

Biological sex and age-related differences shape the antiviral response to SARS-CoV-2 infection.

For the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, clinical manifestations are broad and highly heterogeneous for both sexes. We aimed to determine how biological sex and age impact immune gene expression, particularly influencing the humoral neutralizing antibody (NAb) response and the cytokine production in coronavirus disease 2019 (COVID-19) subjects. The immune gene expression, according to biological sex and age, was assessed using the genome wide expression profile of blood proteins from healthy individuals using the Genotype Tissue Expression (GTEx) database. Moreover, anti-SARS-CoV-2 neutralizing antibody titers and cytokine levels were determined in blood samples from 141 COVID-19 individuals from Medellín, Colombia. Among subjects with COVID-19, males had statistically significantly higher median NAb titers and serum concentrations of interleukin-6 and CC chemokine ligand 3 than females. Overall, our findings point out a more robust innate immune response in women that could help recognize and restrain the virus faster than in men.

Transcriptional and post-transcriptional mechanisms that regulate the genetic program in Zika virus-infected macrophages.

Besides our understanding of the effects of ZIKA virus (ZIKV) infection on neural progenitors' cells the pathogenesis of this RNA virus also involves antigen-presenting cells, including macrophages. However, the molecular mechanisms that control gene activation and repression associated with the macrophage response to acute ZIKV infection are not fully understood. We approached the issue by RNA-seq and miRNA-seq datasets to understand the genetic program of ZIKV-infected macrophages. Results indicate that macrophage activates a regulatory program, involving 1067 differentially expressed genes. These genetic programs induced an inflammatory response mediated by chemokines as well as an interferon-independent anti-viral response, presumptively activated by IL-27. Additionally, the pathogenetic process involves changes in other signaling pathways such as cellular stress, cell signaling, metabolism, and cell differentiation. Furthermore, transcriptional control analysis revealed regulatory functions of key transcription factors principally, NFκB and STAT1, as well as HIF1A, ETV7, and PRMD1 that are associated with metabolic reprogramming during viral infection. We also noted six long-noncoding RNAs (lncRNAs) that may act in the regulation of gene expression, including MROCKI and ZC2HC1A-2, that are involved in the inflammatory response and expression of the cytokines, respectively. On the other hand, post-transcriptional control by miRNAs, including miR-155-5p and miR-146a-5p, are associated with modulation of genes related to inflammatory and antiviral responses. Relevant to the post-transcriptional control, our data unveiled the role of RNA binding proteins that have diverse functions such as ribonucleases (PNPT1, ZC3H12A, and ZC3HAV1), splicing factors (SSB, RBM11, and RAVER2), and RNA modifiers (PARP10 and PARP14). Overall, the results establish an unbiased approach to discerning the wiring of a regulatory mechanism controlling the genetic program in ZIKV-infected macrophages.

Vitamin D boosts immune response of macrophages through a regulatory network of microRNAs and mRNAs.

Vitamin D is associated with the stimulation of innate immunity, inflammation, and host defense against pathogens. Macrophages express receptors of Vitamin D, regulating the transcription of genes related to immune processes. However, the transcriptional and post-transcriptional strategies controlling gene expression in differentiated macrophages, and how they are influenced by Vitamin D are not well understood. We studied whether Vitamin D enhances immune response by regulating the expression of microRNAs and mRNAs. Analysis of the transcriptome showed differences in expression of 199 genes, of which 68% were up-regulated, revealing the cell state of monocyte-derived macrophages differentiated with Vitamin D (D3-MDMs) as compared to monocyte-derived macrophages (MDMs). The differentially expressed genes appear to be associated with pathophysiological processes, including inflammatory responses, and cellular stress. Transcriptional motifs in promoter regions of up- or down-regulated genes showed enrichment of VDR motifs, suggesting possible roles of transcriptional activator or repressor in gene expression. Further, the microRNA-Seq analysis indicated that there were 17 differentially expressed miRNAs, of which, seven were up-regulated and 10 down-regulated, suggesting that Vitamin D plays a critical role in the regulation of miRNA expression during macrophages differentiation. The miR-6501-3p, miR-1273h-5p, miR-665, miR-1972, miR-1183, miR-619-5p were down-regulated in D3-MDMs compared to MDMs. The integrative analysis of miRNA and mRNA expression profiles predicts that miR-1972, miR-1273h-5p, and miR-665 regulate genes PDCD1LG2, IL-1B, and CD274, which are related to the inflammatory response. Results suggest an essential role of Vitamin D in macrophage differentiation that modulates host response against pathogens, inflammation, and cellular stress.

Degree of piRNA sharing and Piwi gene expression in the skeletal muscle of Piaractus mesopotamicus (pacu), Colossoma macropomum (tambaqui), and the hybrid tambacu.

PiRNAs are a class of small noncoding RNAs that, in their mature form, bind to Piwi proteins to repress transposable element activity. Besides their role in gametogenesis and genome integrity, recent evidence indicates their action in non-germinative tissues. We performed a global analysis of piRNA and Piwi gene expression in the skeletal muscle of juveniles pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), and the hybrid tambacu to evaluate the degree of piRNA sharing among these three genotypes. Total RNA was sequenced and analyzed using specific parameters of piRNAs by bioinformatics tools. piRNA and Piwi gene expression was analyzed by RT-qPCR. We detected 24 piRNA clusters common to the three genotypes, with eight shared between pacu and tambacu, three between pacu and tambaqui, and five between tambaqui and tambacu; seven, five, and four clusters were unique to pacu, tambacu, and tambaqui, respectively. Genomic localization and fold change values showed two clusters and 100 piRNAs shared among the three genotypes. The gene expression of four piRNAs was evaluated to validate our bioinformatics results. piRNAs from cluster 17 were higher in tambacu than pacu and piRNAs from cluster 18 were more highly expressed in tambacu than tambaqui and pacu. In addition, the expression of Piwis 1 and 2 was higher in tambacu and tambaqui than pacu. Our results open an important window to investigate whether these small noncoding RNAs benefit the hybrid in terms of faster growth and offer a new perspective on the function of piRNAs and Piwis in fish skeletal muscle.

Vitamin D Regulates the Expression of Immune and Stress Response Genes in Dengue Virus-infected Macrophages by Inducing Specific MicroRNAs.

BACKGROUND: The pathogenesis associated with Dengue virus (DENV) infection is marked by the impairment of host immune response. Consequently, the modulation of immune response has emerged as an important therapeutic target for the control of DENV infection. Vitamin D has been shown to regulate the immune response in DENV infection, although the molecular mechanism remains poorly understood. Post-transcriptional regulation of mRNA by miRNAs offers an opportunity to gain insight into the immunomodulation mediated by vitamin D. OBJECTIVE: Previously, it has been observed that a high dose of vitamin D (4000 IU) decreased DENV-2 infection and inflammatory response in monocyte-derived macrophages (MDMs). Here, we examine whether high or low doses of vitamin D supplements exert differential effect on miRNA expression in DENV-infected macrophages. METHODS: We analyzed miRNA expression profiles in MDMs isolated from healthy individuals who were given either 1000 or 4000 IU/day of vitamin D for 10 days. MDMs before or after vitamin D supplementation were challenged with DENV-2, and miRNAs profiles were analyzed by qPCR arrays. RESULTS: DENV-2 infected MDMs supplemented with 4000 IU, showed up-regulation of miR-374a-5p, miR-363-3p, miR-101-3p, miR-9-5p, miR-34a-5p, miR-200a-3p, and the family of miRNAs miR-21-5p, and miR-590-p. The miRNA profile and predicted target mRNAs suggested regulatory pathways in MDMs obtained from healthy donors who received higher doses of vitamin D. These DENV-2 infected MDMs expressed a unique set of miRNAs that target immune and cellular stress response genes. CONCLUSION: The results suggest vitamin D dose-dependent differential expression of miRNAs target key signaling pathways of the pathogenesis of dengue disease.

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

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.

New LncRNAs in Chronic Hepatitis C progression: from fibrosis to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world, and about 80% of the cases are associated with hepatitis B or C. Genetic and epigenetic alterations are accumulated over decades of chronic injury and may affect the functioning of tumor suppressor genes and protooncogenes. Studies have evidenced the role of Long non-coding RNAs (LncRNA) with oncogenic or tumor suppressor activities, suggesting a great potential in the treatment, diagnosis or indicator of prognosis in cancer. In this context, the aim of this study was to evaluate the global expression profile lncRNA in hepatic tissue samples with different stages of fibrosis associated with chronic hepatitis C, HCC and normal liver, in order to identify new lncRNAs that could contribute to study the progression of hepatic fibrosis to HCC associated with chronic hepatitis C. RNA-Seq was performed on Illumina NextSeq platform to identify lncRNAs expressed differently in 15 patients with chronic hepatitis C, three patients with HCC and three normal liver specimens. When the pathological tissues (fibrosis and carcinoma) were compared to normal hepatic tissue, were identified 2, 6 e 34 differentially expressed lncRNAs in moderate fibrosis, advanced fibrosis and HCC, respectively. The carcinoma group had the highest proportion of differentially expressed lncRNA (34) and of these, 29 were exclusive in this type of tissue. A heat map of the deregulated lncRNA revealed different expression patterns along the progression of fibrosis to HCC. The results showed the deregulation of some lncRNA already classified as tumor suppressors in HCC and other cancers, as well as some unpublished lncRNA whose function is unknown. Some of these lncRNAs are dysregulated since the early stages of liver injury in patients with hepatitis C, others overexpressed only in tumor tissue, indicating themselves as candidates of markers of fibrosis progression or tumor, with potential clinical applications in prognosis as well as a therapeutic target. Although there are already studies on lncRNA in hepatocellular carcinoma, this is the first study conducted in samples exclusively of HCV-related liver and HCV HCC.

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

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 (r2 : 0.80; P value: 0.002) and weight loss (r2 : 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.

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

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 IL-6, CSF3, and IL-8. Also, we identified NCAM1, CNTN1, SCG2, CADM1, IL-8, NPTX1, and APOD as PSCB in the tumor secretome. These PSCB were capable of distinguishing worse and better prognosis (recurrence and survival) in NSCLC patients. IL-8 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, IL-8 expression in NSCLC tissues was associated with worse prognosis, and the recombinant IL-8 was capable of triggering atrophy in C2C12 myotubes.

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

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/Foxo1, miR-27a/Mef2c, miR-27b/Cxcl12, miR-27b/Mef2c, miR-140/Cxcl12, miR-199a/Cav1, and miR-199a/Junb, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting MSTN, CXCL12, and CAMK2B, 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.

Osteoglycin inhibition by microRNA miR-155 impairs myogenesis.

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.

MicroRNA profiling of human primary macrophages exposed to dengue virus identifies miRNA-3614-5p as antiviral and regulator of ADAR1 expression.

BACKGROUND: Due to the high burden of dengue disease worldwide, a better understanding of the interactions between dengue virus (DENV) and its human host cells is of the utmost importance. Although microRNAs modulate the outcome of several viral infections, their contribution to DENV replication is poorly understood. METHODS AND PRINCIPAL FINDINGS: We investigated the microRNA expression profile of primary human macrophages challenged with DENV and deciphered the contribution of microRNAs to infection. To this end, human primary macrophages were challenged with GFP-expressing DENV and sorted to differentiate between truly infected cells (DENV-positive) and DENV-exposed but non-infected cells (DENV-negative cells). The miRNAome was determined by small RNA-Seq analysis and the effect of differentially expressed microRNAs on DENV yield was examined. Five microRNAs were differentially expressed in human macrophages challenged with DENV. Of these, miR-3614-5p was found upregulated in DENV-negative cells and its overexpression reduced DENV infectivity. The cellular targets of miR-3614-5p were identified by liquid chromatography/mass spectrometry and western blot. Adenosine deaminase acting on RNA 1 (ADAR1) was identified as one of the targets of miR-3614-5p and was shown to promote DENV infectivity at early time points post-infection. CONCLUSION/SIGNIFICANCE: Overall, miRNAs appear to play a limited role in DENV replication in primary human macrophages. The miRNAs that were found upregulated in DENV-infected cells did not control the production of infectious virus particles. On the other hand, miR-3614-5p, which was upregulated in DENV-negative macrophages, reduced DENV infectivity and regulated ADAR1 expression, a protein that facilitates viral replication.

Differential microRNA Expression in Fast- and Slow-Twitch Skeletal Muscle of Piaractus mesopotamicus during Growth.

Pacu (Piaractus mesopotamicus) is a Brazilian fish with a high economic value in pisciculture due to its rusticity and fast growth. Postnatal growth of skeletal muscle in fish occurs by hyperplasia and/or hypertrophy, processes that are dependent on the proliferation and differentiation of myoblasts. A class of small noncoding RNAs, known as microRNAs (miRNAs), represses the expression of target mRNAs, and many studies have demonstrated that miR-1, miR-133, miR-206 and miR-499 regulate different processes in skeletal muscle through the mRNA silencing of hdac4 (histone deacetylase 4), srf (serum response factor), pax7 (paired box 7) and sox6 ((sex determining region Y)-box 6), respectively. The aim of our work was to evaluate the expression of these miRNAs and their putative target mRNAs in fast- and slow-twitch skeletal muscle of pacu during growth. We used pacus in three different development stages: larval (aged 30 days), juvenile (aged 90 days and 150 days) and adult (aged 2 years). To complement our study, we also performed a pacu myoblast cell culture, which allowed us to investigate miRNA expression in the progression from myoblast proliferation to differentiation. Our results revealed an inverse correlation between the expression of the miRNAs and their target mRNAs, and there was evidence that miR-1 and miR-206 may regulate the differentiation of myoblasts, whereas miR-133 may regulate the proliferation of these cells. miR-499 was highly expressed in slow-twitch muscle, which suggests its involvement in the specification of the slow phenotype in muscle fibers. The expression of these miRNAs exhibited variations between different development stages and between distinct muscle twitch phenotypes. This work provides the first identification of miRNA expression profiles in pacu skeletal muscle and suggests an important role of these molecules in muscle growth and in the maintenance of the muscle phenotype.