Long Noncoding RNA uc.173 Promotes Renewal of the Intestinal Mucosa by Inducing Degradation of MicroRNA 195.

BACKGROUND AND AIMS: The mammalian intestinal epithelium self-renews rapidly and homeostasis is preserved via tightly controlled mechanisms. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions, but little is known about their role in maintaining the integrity of the intestinal epithelium. We searched for T-UCRs that regulate growth of the intestinal mucosa and investigated the mechanism by which T-UCR uc.173 regulates epithelial renewal. METHODS: C57BL/6J mice were deprived of food for 48 hours in fasting experiments. Some mice were given intraperitoneal injections of a plasmid encoding LNA-anti-uc.173, to knock down endogenous uc.173. For studies using organoids, primary enterocytes were isolated from the intestine and transfected with the uc.173 transgene to increase uc.173 levels. Intestinal epithelial cells (Caco-2 and IEC-6 lines) were transfected with LNA-anti-uc.173 or uc.173 transgene. We quantified intestinal epithelial renewal based on BrdU incorporation, villus height and crypt depth, and cell number. The association of uc.173 with microRNA 195 (miRNA195) was determined by RNA pull-down assays. RESULTS: Genome-wide profile analyses identified 21 T-UCRs, including uc.173, that were differentially expressed between intestinal mucosa of fasted vs non-fasted mice. Increasing levels of uc.173 by expression of a transgene increased growth of intestinal epithelial cells and organoids. Decreasing uc.173 levels by LNA-anti-uc.173 in mice reduced renewal of the intestinal epithelium. We found that uc.173 interacted directly with the primary transcript of miRNA195, leading to miRNA195 degradation. CONCLUSIONS: In analyses of intestinal epithelial cells and mice, we identified uc.173 noncoding RNA that regulates growth of the intestinal mucosa and stimulates intestinal epithelial renewal by reducing levels of miRNA195.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

OBJECTIVE: To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. APPROACH AND RESULTS: In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. CONCLUSIONS: These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Post-transcriptional regulation of Wnt co-receptor LRP6 and RNA-binding protein HuR by miR-29b in intestinal epithelial cells.

MicroRNAs (miRNAs) control gene expression by binding to their target mRNAs for degradation and/or translation repression and are implicated in many aspects of cellular physiology. Our previous study shows that miR-29b acts as a biological repressor of intestinal mucosal growth, but its exact downstream targets remain largely unknown. In the present study, we found that mRNAs, encoding Wnt co-receptor LRP6 (low-density lipoprotein-receptor-related protein 6) and RNA-binding protein (RBP) HuR, are novel targets of miR-29b in intestinal epithelial cells (IECs) and that expression of LRP6 and HuR is tightly regulated by miR-29b at the post-transcriptional level. miR-29b interacted with both Lrp6 and HuR mRNAs via their 3'-UTRs and inhibited LRP6 and HuR expression by destabilizing Lrp6 and HuR mRNAs and repressing their translation. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-29b through a single binding site in the Lrp6 or HuR 3'-UTR, whereas deletion mutation of this site prevented miR-29b-induced repression of LRP6 and HuR expression. Repression of HuR by miR-29b in turn also contributed to miR-29b-induced LRP6 inhibition, since ectopic overexpression of HuR in cells overexpressing miR-29b restored LRP6 expression to near normal levels. Taken together, our results suggest that miR-29b inhibits expression of LRP6 and HuR post-transcriptionally, thus playing a role in the regulation of IEC proliferation and intestinal epithelial homoeostasis.

The Zinc Finger Transcription Factor BbCmr1 Regulates Conidium Maturation in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn2Cys6 transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H2O2, Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1, and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

Poor consistency between reflux symptom index and laryngopharyngeal pH monitoring in laryngopharyngeal reflux diagnosis in Chinese population.

BACKGROUND: It is unknown whether the reflux symptom index (RSI) can replace pH monitoring as a diagnostic tool for laryngopharyngeal reflux (LPR) in Chinese people. The relationships between reflux parameters and LPR symptoms also require further research. METHODS: A total of 216 Chinese patients underwent laryngopharyngeal pH monitoring and filled out an RSI questionnaire. Laryngopharyngeal pH monitoring indicated a diagnosis of LPR for patients with 7 or more episodes of reflux or a reflex area index (RAI) of 6.3 or more. The RSI questionnaire indicated a diagnosis of LPR for patients with RSI scores of 14 or higher. RESULTS: Of the 216 patients, 85 were diagnosed with LPR as assessed by the RSI, and 72 were diagnosed with LPR through laryngopharyngeal pH monitoring. The Cohen's kappa coefficient comparing LPR diagnosis consistency between RSI score and laryngopharyngeal pH monitoring was 0.133 (P=0.007). This indicated the two diagnostic methods were consistent to a low degree; the total consistency rate was only 59.7% (129/216). The sensitivity of the RSI was 48.6% (35/72), and its specificity was 82.5% (94/114). For convenience, we named the nine symptom groups in the RSI sequentially as P1-P9. P1, P2, P3, P5, P6, and P7 were all correlated with at least one reflux parameter (P<0.05), but P4, P8, and P9 were not correlated with any reflux parameters (P>0.05). A total of 72 patients were diagnosed using pH monitoring, the gold standard for LPR diagnosis. The most common symptoms of LPR were found to be P9, P3, P8, P7, and P2 in these patients. The symptoms that most seriously affected patients were P9, P8, P3, P7, and P2. CONCLUSIONS: The consistency in diagnosis of LPR between the RSI and laryngopharyngeal pH monitoring was poor, meaning the RSI is not a suitable LPR initial screening tool and cannot replace pH monitoring. Additionally, reflux symptoms P4, P8, and P9 were not correlated with any reflux parameters. The most prevalent LPR symptom was P9, followed by P3, P8, P7, and P2. The most severe symptom was also P9, followed by P8, P3, P7, and P2.

Oral vaccination with attenuated Salmonella encoding the Trichinella spiralis 43-kDa protein elicits protective immunity in BALB/c mice.

A vaccine against Trichinella spiralis infection is urgently needed to interrupt its transmission from domestic animals to humans. However, no vaccine against T. spiralis is currently available. Our previous study demonstrated that the use of the 43-kDa glycoprotein present in excretory-secretory (ES) proteins of muscle larvae (ML) as an intramuscular DNA vaccine led to a 52.1% protection rate against T. spiralis infection. Attenuated Salmonella strains have the advantage of eliciting mucosal immunity, which is important for controlling T. spiralis infections at the intestinal stage and can be provided as vaccines via oral or intranasal routes. Therefore, in this study, complete 43-kDa glycoprotein (Ts43) sequences of T. spiralis were cloned into the vector pYA3681, and the recombinant plasmid pYA3681-Ts43 was transformed into the attenuated Salmonella typhimurium strain χ11802. The results showed that oral vaccination of mice with attenuated Salmonella carrying the recombinant plasmid pYA3681-Ts43 induced an evident elevation of the local intestinal mucosal sIgA and serum IgG antibody responses. The flow cytometry results showed that the percentages of CD4+ T cells and secreted IFN-γ, IL-4, and IL-17A in CD4+ T cells were significantly increased in the spleen and mesenteric lymph node (MLN) lymphocytes of the vaccinated groups. In addition, increased levels of the IFN-γ, IL-4, and IL-17A cytokines were also observed in the serum of the immunized groups. The above immune response results in the immunized groups demonstrated that protective immunity was elicited in this study. Finally, vaccinated mice demonstrated a significant 45.9% reduction in ML burden after infection with T. spiralis. This study demonstrated that oral vaccination with Ts43 delivered by attenuated Salmonella elicited local and systemic concurrent Th1/Th2/Th17 immune responses and provided partial protection against T. spiralis infection in BALB/c mice. This is a prospective strategy for the prevention and control of trichinellosis.

Role of flow-sensitive microRNAs and long noncoding RNAs in vascular dysfunction and atherosclerosis.

Atherosclerosis is the primary underlying cause of myocardial infarction, ischemic stroke, and peripheral artery disease. The disease preferentially occurs in arterial regions exposed to disturbed blood flow, in part, by altering expression of flow-sensitive coding- and non-coding genes. In this review, we summarize the role of noncoding RNAs, [microRNAs (miRNAs) and long noncoding RNAs(lncRNAs)], as regulators of gene expression and outline their relationship to the pathogenesis of atherosclerosis. While miRNAs are small noncoding genes that post-transcriptionally regulate gene expression by targeting mRNA transcripts, the lncRNAs regulate gene expression by diverse mechanisms, which are still emerging and incompletely understood. We focused on multiple flow-sensitive miRNAs such as, miR-10a, -19a, -23b, -17~92, -21, -663, -92a, -143/145, -101, -126, -712, -205, and -155 that play a critical role in endothelial function and atherosclerosis by targeting inflammation, cell cycle, proliferation, migration, apoptosis, and nitric oxide signaling. Flow-dependent regulation of lncRNAs is just emerging, and their role in vascular dysfunction and atherosclerosis is unknown. Here, we discuss the flow-sensitive lncRNA STEEL along with other lncRNAs studied in the context of vascular pathophysiology and atherosclerosis such as MALAT1, MIAT1, ANRIL, MYOSLID, MEG3, SENCR, SMILR, LISPR1, and H19. Also discussed is the use of these noncoding RNAs as potential biomarkers and therapeutics to reduce and regress atherosclerosis.

Circulating miR-182-5p and miR-5187-5p as biomarkers for the diagnosis of unprotected left main coronary artery disease.

BACKGROUND: Patients with unprotected left main coronary artery disease (uLMCAD) have high mortality rate due to sudden heart failure and acute myocardial infarction, for which reliable diagnostic biomarkers to detect this disease at an early stage are in urgent need. Circulating microRNAs (miRNAs) have emerged as a class of novel biomarkers for cardiovascular diseases. The purpose of this study was to investigate utility of miRNAs as biomarkers for early detection of uLMCAD. METHODS: High-throughput sequencing (NGS) was initially employed to compare circulating miRNA expression profiles in uLMCAD patients to that in patients without coronary artery disease (CAD) to identify candidate miRNA biomarkers. We further validated the expression of candidate miRNAs by quantitative polymerase chain reaction (qPCR) in a larger cohort. Receiver operating characteristic (ROC) analysis with multivariate logistic regression was used to evaluate the diagnostic power of candidate miRNAs individually and combined. RESULTS: MiR-182-5p, miR-199a-5p and miR-5187-5p were found significantly differentially expressed through NGS (fold changes =1.35, 1.65, 0.5, P values =0.018, 0.046, 0.030, respectively, n=5 for both uLMCAD group and non-CAD control group). In a larger cohort (n=27 for uLMCAD patient and n=38 for non-CAD controls), qPCR confirmed that expression of miR-182-5p was up-regulated (2.57-fold, P=0.011) and expression of miR-5187-5p was down-regulated (0.47-fold, P=0.018) in the plasma of uLMCAD patients. ROC analysis with multivariate logistic regression show that miR-182 and miR-5187 have an AUC score of 0.97 and 0.94 respectively, indicating high diagnostic power as biomarkers for uLMCAD. Interestingly, correlation analysis suggests that the expression of two miRNAs were independent to each other. CONCLUSIONS: These results suggested that circulating miR-182-5p and miR-5187-5p were suitable diagnostic biomarkers for uLMCAD, both potentially providing diagnostic information for discriminating uLMCAD patients from non-CAD population prior to invasive diagnostic coronary angiography (CAG).

Regulation of Intestinal Epithelial Barrier Function by Long Noncoding RNA uc.173 through Interaction with MicroRNA 29b.

The mammalian intestinal epithelium establishes a selectively permeable barrier that supports nutrient absorption and prevents intrusion by noxious luminal substances and microbiota. The effectiveness and integrity of the barrier function are tightly regulated via well-controlled mechanisms. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control diverse cellular processes, but their roles in the regulation of gut permeability remain largely unknown. Here we report that the T-UCR uc.173 enhances intestinal epithelial barrier function by antagonizing microRNA 29b (miR-29b). Decreasing the levels of uc.173 by gene silencing led to dysfunction of the intestinal epithelial barrier in cultured cells and increased the vulnerability of the gut barrier to septic stress in mice. uc.173 specifically stimulated translation of the tight junction (TJ) claudin-1 (CLDN1) by associating with miR-29b rather than by binding directly to CLDN1 mRNA. uc.173 acted as a natural decoy RNA for miR-29b, which interacts with CLDN1 mRNA via the 3' untranslated region and represses its translation. Ectopically expressed uc.173 abolished the association of miR-29b with CLDN1 mRNA and restored claudin-1 expression to normal levels in cells overexpressing miR-29b, thus rescuing the barrier function. These results highlight a novel function of uc.173 in controlling gut permeability and define a mechanism by which uc.173 stimulates claudin-1 translation, by decreasing the availability of miR-29b to CLDN1 mRNA.

Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.

Exosomes (Exo) secreted from hypoxia-conditioned bone marrow mesenchymal stem cells (BM-MSCs) were found to be protective for ischemic disease. However, the role of exosomal miRNA in the protective effect of hypoxia-conditioned BM-MSCs-derived Exo (Hypo-Exo) remains largely uncharacterized and the poor specificity of tissue targeting of Exo limits their clinical applications. Therefore, the objective of this study was to examine the effect of miRNA in Hypo-Exo on the repair of ischemic myocardium and its underlying mechanisms. We further developed modified Hypo-Exo with high specificity to the myocardium and evaluate its therapeutic effects. Methods: Murine BM-MSCs were subjected to hypoxia or normoxia culture and Exo were subsequently collected. Hypo-Exo or normoxia-conditioned BM-MSC-derived Exo (Nor-Exo) were administered to mice with permanent condition of myocardial infarction (MI). After 28 days, to evaluate the therapeutic effects of Hypo-Exo, infarction area and cardio output in Hypo-Exo and Nor-Exo treated MI mice were compared through Masson's trichrome staining and echocardiography respectively. We utilized the miRNA array to identify the significantly differentially expressed miRNAs between Nor-Exo and Hypo-Exo. One of the most enriched miRNA in Hypo-Exo was knockdown by applying antimiR in Hypoxia-conditioned BM-MSCs. Then we performed intramyocardial injection of candidate miRNA-knockdown-Hypo-Exo in a murine MI model, changes in the candidate miRNA's targets expression of cardiomyocytes and the cardiac function were characterized. We conjugated Hypo-Exo with an ischemic myocardium-targeted (IMT) peptide by bio-orthogonal chemistry, and tested its targeting specificity and therapeutic efficiency via systemic administration in the MI mice. Results: The miRNA array revealed significant enrichment of miR-125b-5p in Hypo-Exo compared with Nor-Exo. Administration of miR-125b knockdown Hypo-Exo significantly increased the infarction area and suppressed cardiomyocyte survival post-MI. Mechanistically, miR-125b knockdown Hypo-Exo lost the capability to suppress the expression of the proapoptotic genes p53 and BAK1 in cardiomyocytes. Intravenous administration of IMT-conjugated Hypo-Exo (IMT-Exo) showed specific targeting to the ischemic lesions in the injured heart and exerted a marked cardioprotective function post-MI. Conclusion: Our results illustrate a new mechanism by which Hypo-Exo-derived miR125b-5p facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. Furthermore, our IMT- Exo may serve as a novel drug carrier that enhances the specificity of drug delivery for ischemic disease.

Posttranscriptional regulation of intestinal epithelial integrity by noncoding RNAs.

Maintenance of the gut epithelial integrity under stressful environments requires epithelial cells to rapidly elicit changes in gene expression patterns to regulate their survival, adapt to stress, and keep epithelial homeostasis. Disruption of the intestinal epithelial integrity occurs commonly in patients with various critical illnesses, leading to the translocation of luminal toxic substances and bacteria to the blood stream. Recently, noncoding RNAs (ncRNAs) have emerged as a novel class of master regulators of gene expression and are fundamentally involved in many aspects of gut mucosal regeneration, protection, and epithelial barrier function. Here, we highlight the roles of several intestinal epithelial tissue-specific microRNAs, including miR-222, miR-29b, miR-503, and miR-195, and long ncRNAs such as H19 and SPRY4-IT1 in the regulation of cell proliferation, apoptosis, migration, and cell-to-cell interactions and also further analyze the mechanisms through which ncRNAs and their interactions with RNA-binding proteins modulate the stability and translation of target mRNAs. WIREs RNA 2017, 8:e1399. doi: 10.1002/wrna.1399 For further resources related to this article, please visit the WIREs website.

H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR.

The disruption of the intestinal epithelial barrier function occurs commonly in various pathologies, but the exact mechanisms responsible are unclear. The H19 long noncoding RNA (lncRNA) regulates the expression of different genes and has been implicated in human genetic disorders and cancer. Here, we report that H19 plays an important role in controlling the intestinal epithelial barrier function by serving as a precursor for microRNA 675 (miR-675). H19 overexpression increased the cellular abundance of miR-675, which in turn destabilized and repressed the translation of mRNAs encoding tight junction protein ZO-1 and adherens junction E-cadherin, resulting in the dysfunction of the epithelial barrier. Increasing the level of the RNA-binding protein HuR in cells overexpressing H19 prevented the stimulation of miR-675 processing from H19, promoted ZO-1 and E-cadherin expression, and restored the epithelial barrier function to a nearly normal level. In contrast, the targeted deletion of HuR in intestinal epithelial cells enhanced miR-675 production in the mucosa and delayed the recovery of the gut barrier function after exposure to mesenteric ischemia/reperfusion. These results indicate that H19 interacts with HuR and regulates the intestinal epithelial barrier function via the H19-encoded miR-675 by altering ZO-1 and E-cadherin expression posttranscriptionally.

Ex vivo nicotine stimulation augments the efficacy of human peripheral blood mononuclear cell-derived dendritic cell vaccination via activating Akt-S6 pathway.

Our previous studies showed that α7 nicotinic acetylcholine receptor (nAchR) agonist nicotine has stimulatory effects on murine bone marrow-derived semimature DCs, but the effect of nicotine on peripheral blood mononuclear cell- (PBMC-) derived human semimature dendritic cells (hu-imDCs) is still to be clarified. In the present study, hu-imDCs (cultured 4 days) were conferred with ex vivo lower dose nicotine stimulation and the effect of nicotine on surface molecules expression, the ability of cross-presentation, DCs-mediated PBMC priming, and activated signaling pathways were determined. We could demonstrate that the treatment with nicotine resulted in increased surface molecules expression, enhanced hu-imDCs-mediated PBMC proliferation, upregulated release of IL-12 in the supernatant of cocultured DCs-PBMC, and augmented phosphorylation of Akt and ribosomal protein S6. Nicotine associated with traces of LPS efficiently enhanced endosomal translocation of internalized ovalbumin (OVA) and increased TAP-OVA colocalization. Importantly, the upregulation of nicotine-increased surface molecules upregulation was significantly abrogated by the inhibition of Akt kinase. These findings demonstrate that ex vivo nicotine stimulation augments hu-imDCs surface molecules expression via Akt-S6 pathway, combined with increased Ag-presentation result in augmented efficacy of DCs-mediated PBMC proliferation and Th1 polarization.