Systematic transcriptome wide analysis of lncRNA-miRNA interactions.

BACKGROUND: Long noncoding RNAs (lncRNAs) are a recently discovered class of non-protein coding RNAs, which have now increasingly been shown to be involved in a wide variety of biological processes as regulatory molecules. The functional role of many of the members of this class has been an enigma, except a few of them like Malat and HOTAIR. Little is known regarding the regulatory interactions between noncoding RNA classes. Recent reports have suggested that lncRNAs could potentially interact with other classes of non-coding RNAs including microRNAs (miRNAs) and modulate their regulatory role through interactions. We hypothesized that lncRNAs could participate as a layer of regulatory interactions with miRNAs. The availability of genome-scale datasets for Argonaute targets across human transcriptome has prompted us to reconstruct a genome-scale network of interactions between miRNAs and lncRNAs. RESULTS: We used well characterized experimental Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) datasets and the recent genome-wide annotations for lncRNAs in public domain to construct a comprehensive transcriptome-wide map of miRNA regulatory elements. Comparative analysis revealed that in addition to targeting protein-coding transcripts, miRNAs could also potentially target lncRNAs, thus participating in a novel layer of regulatory interactions between noncoding RNA classes. Furthermore, we have modeled one example of miRNA-lncRNA interaction using a zebrafish model. We have also found that the miRNA regulatory elements have a positional preference, clustering towards the mid regions and 3' ends of the long noncoding transcripts. We also further reconstruct a genome-wide map of miRNA interactions with lncRNAs as well as messenger RNAs. CONCLUSIONS: This analysis suggests widespread regulatory interactions between noncoding RNAs classes and suggests a novel functional role for lncRNAs. We also present the first transcriptome scale study on miRNA-lncRNA interactions and the first report of a genome-scale reconstruction of a noncoding RNA regulatory interactome involving lncRNAs

Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication

protein expression and HIV-1 replicatio

Human embryonic stem cell-derived endothelial cell product injection attenuates cardiac remodeling in myocardial infarction

BACKGROUND: Mechanisms contributing to tissue remodeling of the infarcted heart following cell-based therapy remain elusive. While cell-based interventions have the potential to influence the cardiac healing process, there is little direct evidence of preservation of functional myocardium. AIM: The aim of the study was to investigate tissue remodeling in the infarcted heart following human embryonic stem cell-derived endothelial cell product (hESC-ECP) therapy. MATERIALS AND METHODS: Following coronary artery ligation (CAL) to induce cardiac ischemia, we investigated infarct size at 1 day post-injection in media-injected controls (CALM, n = 11), hESC-ECP-injected mice (CALC, n = 10), and dead hESC-ECP-injected mice (CALD, n = 6); echocardiography-based functional outcomes 14 days post-injection in experimental (CALM, n = 13; CALC, n = 17) and SHAM surgical mice (n = 4); and mature infarct size (CALM and CALC, both n = 6). We investigated ligand–receptor interactions (LRIs) in hESC-ECP cell populations, incorporating a publicly available C57BL/6J mouse cardiomyocyte-free scRNAseq dataset with naive, 1 day, and 3 days post-CAL hearts. RESULTS: Human embryonic stem cell-derived endothelial cell product injection reduces the infarct area (CALM: 54.5 ± 5.0%, CALC: 21.3 ± 4.9%), and end-diastolic (CALM: 87.8 ± 8.9 uL, CALC: 63.3 ± 2.7 uL) and end-systolic ventricular volume (CALM: 56.4 ± 9.3 uL, CALC: 33.7 ± 2.6 uL). LRI analyses indicate an alternative immunomodulatory effect mediated via viable hESC-ECP-resident signaling. CONCLUSION: Delivery of the live hESC-ECP following CAL modulates the wound healing response during acute pathological remodeling, reducing infarct area, and preserving functional myocardium in this relatively acute model. Potential intrinsic myocardial cellular/hESC-ECP interactions indicate that discreet immunomodulation could provide novel therapeutic avenues to improve cardiac outcomes following myocardial infarction

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 (gib005Δ8/+)) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability

CARMN loss regulates smooth muscle cells and accelerates atherosclerosis in mice

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Forward genetic screen using a gene-breaking trap approach identifies a novel role of grin2bb-associated RNA transcript (grin2bbART) in zebrafish heart function

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it “bigheart.” Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish

Antagonism of microRNA function in zebrafish embryos by using Locked Nucleic Acid enzymes (LNAzymes)

MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos

Depicting lncRNA transcripts targeted by miRNAs.

<p>The figure shows the lncRNA transcripts targeted by miRNAs along with the distribution of Ago types in the targeting complex. The presence and absence of specific Ago type in the RISC complex is represented by the difference in the colors in the heatmap. The targeting miRNA has also been mentioned.</p

Binding alignments of microRNAs targeting predicted zebrafish lncRNA.

<p>The figure shows predicted binding alignment of miR-125a, miR-125b, miR-125c, miR-17a*, miR-20*, miR-210*, miR-29a, miR-29b and miR457a with predicted zebrafish lncRNA.</p