MiRNAs with Apoptosis Regulating Potential Are Differentially Expressed in Chronic Exercise-Induced Physiologically Hypertrophied Hearts

Physiological cardiac hypertrophy is an adaptive mechanism, induced during chronic exercise. As it is reversible and not associated with cardiomyocyte death, it is considered as a natural tactic to prevent cardiac dysfunction and failure. Though, different studies revealed the importance of microRNAs (miRNAs) in pathological hypertrophy, their role during physiological hypertrophy is largely unexplored. Hence, this study is aimed at revealing the global expression profile of miRNAs during physiological cardiac hypertrophy. Chronic swimming protocol continuously for eight weeks resulted in induction of physiological hypertrophy in rats and histopathology revealed the absence of tissue damage, apoptosis or fibrosis. Subsequently, the total RNA was isolated and small RNA sequencing was executed. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during physiological hypertrophy. The expression profile of the significantly differentially expressed miRNAs was validated by qPCR. In silico prediction of target genes by miRanda, miRdB and TargetScan and subsequent qPCR analysis unraveled that miRNAs including miR-99b, miR-100, miR-19b, miR-10, miR-208a, miR-133, miR-191a, miR-22, miR-30e and miR-181a are targeting the genes that primarily regulate cell proliferation and cell death. Gene ontology and pathway mapping showed that the differentially expressed miRNAs and their target genes were mapped to apoptosis and cell death pathways principally via PI3K/Akt/mTOR and MAPK signaling. In summary, our data indicates that regulation of these miRNAs with apoptosis regulating potential can be one of the major key factors in determining pathological or physiological hypertrophy by controlling fibrosis, apoptosis and cell death mechanisms.     

Novel transcriptome data analysis implicates circulating microRNAs in epigenetic inheritance in mammals

Experimental evidence supports a role of mobile small non-coding RNAs in mediating soma to germline hereditary information transfer in epigenetic inheritance in plants and worms. Similar evidence in mammals has not been reported so far. In this bioinformatic analysis, differentially expressed microRNAs (miRNAs) or mRNAs reported previously in genome level expression profiling studies related to or relevant in epigenetic inheritance in mammals were examined for circulating miRNA association. The reported sets of differentially expressed miRNAs or miRNAs that are known to target the reported sets of differentially expressed genes, in that order, showed enrichment of circulating miRNAs across environmental factors, tissues, life cycle stages, generations, genders and species. Circulating miRNAs commonly representing the expression profiles enriched various epigenetic processes. These results provide bioinformatic evidence for a role of circulating miRNAs in epigenetic inheritance in mammals.     

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and Results

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.     

miRNA Profiling Reveals Dysregulation of RET and RET-Regulating Pathways in Hirschsprung's Disease

Hirschsprung’s disease (HSCR), the most common congenital malformation of the gut, is regulated by multiple signal transduction pathways. Several components of these pathways are important targets for microRNAs (miRNAs). Multiple miRNAs have been associated with the pathophysiology of HSCR, and serum miRNAs profiles of HSCR patients have been reported, but miRNA expression in HSCR colon tissue is almost completely unexplored. Using microarray technology, we screened colon tissue to detect miRNAs whose expression profiles were altered in HSCR and identify targets of differentially expressed miRNAs. Following filtering of low-intensity signals, data normalization, and volcano plot filtering, we identified 168 differentially expressed miRNAs (104 up-regulated and 64 down-regulated). Fifty of these mRNAs represent major targets of dysegulated miRNAs and may thus important roles in the pathophysiology of HSCR. Pathway analysis revealed that 7 of the miRNA targets encode proteins involved in regulation of cell proliferation and migration via RET and related signaling pathways (MAPK and PI3K/AKT). Our results identify miRNAs that play key roles in the pathophysiology of the complex multi-factorial disease HSCR.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.     

Type 2 Diabetes Monocyte MicroRNA and mRNA Expression: Dyslipidemia Associates with Increased Differentiation-Related Genes but Not Inflammatory Activation

There is increasing evidence that inflammatory macrophages in adipose tissue are involved in insulin resistance of type 2 diabetes (T2D). Due to a relative paucity of data on circulating monocytes in T2D, it is unclear whether the inflammatory changes of adipose tissue macrophages are reflected in these easily accessible cells.

Objective

To study the expression pattern of microRNAs and mRNAs related to inflammation in T2D monocytes.

Design

A microRNA finding study on monocytes of T2D patients and controls using array profiling was followed by a quantitative Real Time PCR (qPCR) study on monocytes of an Ecuadorian validation cohort testing the top over/under-expressed microRNAs. In addition, monocytes of the validation cohort were tested for 24 inflammation-related mRNAs and 2 microRNAs previously found deregulated in (auto)-inflammatory monocytes.

Results

In the finding study, 142 significantly differentially expressed microRNAs were identified, 15 having the strongest power to discriminate T2D patients from controls (sensitivity 66%, specificity 90%). However, differences in expression of these microRNAs between patients and controls were small. On the basis of >1.4 or <0.6-fold change expression 5 microRNAs were selected for further validation. One microRNA (miR-34c-5p) was validated as significantly over-expressed in T2D monocytes. In addition, we found over expression of 3 mRNAs (CD9, DHRS3 and PTPN7) in the validation cohort. These mRNAs are important for cell morphology, adhesion, shape change, and cell differentiation. Classical inflammatory genes (e.g. TNFAIP3) were only over-expressed in monocytes of patients with normal serum lipids. Remarkably, in dyslipidemia, there was a reduction in the expression of inflammatory genes (e.g. ATF3, DUSP2 and PTGS2).

Conclusions

The expression profile of microRNAs/mRNAs in monocytes of T2D patients indicates an altered adhesion, differentiation, and shape change potential. Monocyte inflammatory activation was only found in patients with normal serum lipids. Abnormal lipid values coincided with a reduced monocyte inflammatory state.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Comprehensive Dissection of PDGF-PDGFR Signaling Pathways in PDGFR Genetically Defined Cells

Despite the growing understanding of PDGF signaling, studies of PDGF function have encountered two major obstacles: the functional redundancy of PDGFRα and PDGFRβ in vitro and their distinct roles in vivo. Here we used wild-type mouse embryonic fibroblasts (MEF), MEF null for either PDGFRα, β, or both to dissect PDGF-PDGFR signaling pathways. These four PDGFR genetically defined cells provided us a platform to study the relative contributions of the pathways triggered by the two PDGF receptors. They were treated with PDGF-BB and analyzed for differential gene expression, in vitro proliferation and differential response to pharmacological effects. No genes were differentially expressed in the double null cells, suggesting minimal receptor-independent signaling. Protean differentiation and proliferation pathways are commonly regulated by PDGFRα, PDGFRβ and PDGFRα/β while each receptor is also responsible for regulating unique signaling pathways. Furthermore, some signaling is solely modulated through heterodimeric PDGFRα/β.     

Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA.

This report presents computational methods of analysis of cellular processes, functions, and pathways affected by differentially expressed microRNA, a statistical basis of the gene enrichment analysis method, a modification of enrichment analysis method accounting for combinatorial targeting of Gene Ontology categories by multiple miRNAs and examples of the global functional profiling of predicted targets of differentially expressed miRNAs in cancer. We have also summarized an application of Ingenuity Pathway Analysis tools for in depth analysis of microRNA target sets that may be useful for the biological interpretation of microRNA profiling data. To illustrate the utility of these methods, we report the main results of our recent computational analysis of five published datasets of aberrantly expressed microRNAs in five human cancers (pancreatic cancer, breast cancer, colon cancer, lung cancer, and lymphoma). Using a combinatorial target prediction algorithm and statistical enrichment analysis, we have determined Gene Ontology categories as well as biological functions, disease categories, toxicological categories, and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in specific cancers. Our recent computational analysis of predicted targets of co-expressed miRNAs in five human cancers suggests that co-expressed miRNAs provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways reportedly affected in a particular cancer.     

The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.     

Analysis of microRNA expression profile in specific pathogen-free chickens in response to reticuloendotheliosis virus infection

Reticuloendotheliosis virus (REV) is an avian retrovirus that causes immunosuppression, growth retardation, and oncogenesis in a variety of birds. REV infection is epidemic in many countries. In this study, we used high-throughput sequencing to identify microRNAs (miRNAs) associated with REV infection. A total of 88 differentially expressed miRNAs were identified in samples collected on days 21 and 28 post-REV infection. Possible target genes of the differentially expressed miRNAs were analyzed. We observed that expression of proapoptotic, proto-oncogene, and carcinogenic cytokine mRNAs was highly upregulated, whereas expression of antiapoptotic cytokine mRNAs was significantly downregulated. Our findings provide a potential link between miRNA expression and the pathogenesis of REV infection.

     

Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.