Kang

miR-210 has an antiapoptotic effect in pulmonary artery smooth muscle cells during hypoxia

MicroRNAs (miRNAs) were recently reported to play an important role in the pathogenesis of pulmonary arterial hypertension (PAH), but it is not clear which miRNAs are important or what pathways are involved in the process. Because hypoxia is an important stimulus for human pulmonary artery smooth muscle cell (HPASMC) proliferation and PAH, we performed miRNA microarray assays in hypoxia-treated and control HPASMC. We found that miR-210 is the predominant miRNA induced by hypoxia in HPASMC. Induction of miR-210 was also observed in whole lungs of mice with chronic hypoxia-induced PAH. We found that transcriptional induction of miR-210 in HPASMC is hypoxia-inducible factor-1α dependent. Inhibition of miR-210 in HPASMC caused a significant decrease in cell number due to increased apoptosis. We found that miR-210 appears to mediate its antiapoptotic effects via the regulation of transcription factor E2F3, a direct target of miR-210. Our results have identified miR-210 as a hypoxia-inducible miRNA both in vitro and in vivo, which inhibits pulmonary vascular smooth muscle cell apoptosis in hypoxia by specifically repressing E2F3 expression.

MicroRNA-124 Suppresses the Transactivation of Nuclear Factor of Activated T Cells by Targeting Multiple Genes and Inhibits the Proliferation of Pulmonary Artery Smooth Muscle Cells

Background: The NFAT signaling pathway is linked to pulmonary arterial hypertension. Results: MicroRNA screening revealed that miR-124 robustly inhibits NFAT activity, dephosphorylation, and nuclear translocation of NFAT by targeting multiple genes, NFATc1, CAMTA1, and PTBP1. Conclusion: miR-124 is an effective and multipronged inhibitor of NFAT signaling. Significance: miR-124 might be a potential immunosuppressant that may have biological effects linked to pulmonary arterial hypertension. Abnormal proliferation and phenotypic modulation of pulmonary artery smooth muscle cells (PASMC) contributes to the pathogenesis of numerous cardiovascular disorders, including pulmonary arterial hypertension (PAH). The nuclear factor of activated T cells (NFAT) signaling pathway is linked to PASMC proliferation and PAH. MicroRNAs (miRNAs) are small non-coding RNAs that function in diverse biological processes. To systemically identify the specific miRNAs that regulate the NFAT pathway, a human primary miRNA library was applied for cell-based high throughput screening with the NFAT luciferase reporter system. Eight miRNAs were found to modulate NFAT activity efficiently. Of them, miR-124 robustly inhibited NFAT reporter activity and decreased both the dephosphorylation and the nuclear translocation of NFAT. miR-124 also inhibited NFAT-dependent transcription of IL-2 in Jurkat T cells. miR-124 exerted its effects by targeting multiple genes, including a known component of the NFAT pathway, NFATc1, and two new regulators of NFAT signaling, CAMTA1 (calmodulin-binding transcription activator 1) and PTBP1 (polypyrimidine tract-binding protein 1). Physiologically, miR-124 was down-regulated by hypoxia in human PASMC, consistent with the activation of NFAT during this process. Down-regulation of miR-124 was also observed in 3-week hypoxia-treated mouse lungs. Furthermore, the overexpression of miR-124 not only inhibited human PASMC proliferation but also maintained its differentiated phenotype by repressing the NFAT pathway. Taken together, our data provide the first evidence that miR-124 acts as an inhibitor of the NFAT pathway. Down-regulation of miR-124 in hypoxia-treated PASMC and its antiproliferative and prodifferentiation effects imply a potential value for miR-124 in the treatment of PAH.

A Novel Real-Time PCR Assay of microRNAs Using S-Poly(T), a Specific Oligo(dT) Reverse Transcription Primer with Excellent Sensitivity and Specificity

Background MicroRNAs (miRNAs) are small, non-coding RNAs capable of postranscriptionally regulating gene expression. Accurate expression profiling is crucial for understanding the biological roles of miRNAs, and exploring them as biomarkers of diseases. Methodology/Principal Findings A novel, highly sensitive, and reliable miRNA quantification approach,termed S-Poly(T) miRNA assay, is designed. In this assay, miRNAs are subjected to polyadenylation and reverse transcription with a S-Poly(T) primer that contains a universal reverse primer, a universal Taqman probe, an oligo(dT)11 sequence and six miRNA-specific bases. Individual miRNAs are then amplified by a specific forward primer and a universal reverse primer, and the PCR products are detected by a universal Taqman probe. The S-Poly(T) assay showed a minimum of 4-fold increase in sensitivity as compared with the stem-loop or poly(A)-based methods. A remarkable specificity in discriminating among miRNAs with high sequence similarity was also obtained with this approach. Using this method, we profiled miRNAs in human pulmonary arterial smooth muscle cells (HPASMC) and identified 9 differentially expressed miRNAs associated with hypoxia treatment. Due to its outstanding sensitivity, the number of circulating miRNAs from normal human serum was significantly expanded from 368 to 518. Conclusions/Significance With excellent sensitivity, specificity, and high-throughput, the S-Poly(T) method provides a powerful tool for miRNAs quantification and identification of tissue- or disease-specific miRNA biomarkers.

Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

MicroRNAs (miRNAs) are small noncoding RNAs (18-25 nucleotides) that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR)-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1) sample collection and preparation; (2) global miRNAs profiling using quantitative real-time PCR (qRT-PCR); (3) data normalization and analysis; and (4) selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

Identification of microRNA-Like RNAs in the Filamentous Fungus Trichoderma reesei by Solexa Sequencing

microRNAs (miRNAs) are non-coding small RNAs (sRNAs) capable of negatively regulating gene expression. Recently, microRNA-like small RNAs (milRNAs) were discovered in several filamentous fungi but not yet in Trichoderma reesei, an industrial filamentous fungus that can secrete abundant hydrolases. To explore the presence of milRNA in T. reesei and evaluate their expression under induction of cellulose, two T. reesei sRNA libraries of cellulose induction (IN) and non-induction (CON) were generated and sequenced using Solexa sequencing technology. A total of 726 and 631 sRNAs were obtained from the IN and CON samples, respectively. Global expression analysis showed an extensively differential expression of sRNAs in T. reesei under the two conditions. Thirteen predicted milRNAs were identified in T. reesei based on the short hairpin structure analysis. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. Computational analysis predicted a number of potential targets relating to many processes including regulation of enzyme expression. The presence and differential expression of T. reesei milRNAs imply that milRNA might play a role in T. reesei growth and cellulase induction. This work lays foundation for further functional study of fungal milRNAs and their industrial application.

An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.

We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive, and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 μl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers.

Hypoxia inducible factor-1 mediates expression of miR-322: potential role in proliferation and migration of pulmonary arterial smooth muscle cells

There is growing evidence that microRNAs play important roles in cellular responses to hypoxia and in pulmonary hypertensive vascular remodeling, but the exact molecular mechanisms involved are not fully elucidated. In this study, we identified miR-322 as one of the microRNAs induced in lungs of chronically hypoxic mice and rats. The expression of miR-322 was also upregulated in primary cultured rat pulmonary arterial smooth muscle cells (PASMC) in response to hypoxia. We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia. Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration. Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration. Our study implicates miR-322 in the hypoxic proliferative response of PASMCs suggesting that it may be playing a role in pulmonary vascular remodeling associated with pulmonary hypertension.

Mulberry fruit prevents LPS-induced NF-κB/pERK/MAPK signals in macrophages and suppresses acute colitis and colorectal tumorigenesis in mice

Here, we investigated the impact of mulberry fruit (MBF) extracts on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages, and the therapeutic efficacy of MBF diet in mice with dextran sulfate sodium (DSS)-induced acute colitis and MUC2−/− mice with colorectal cancer. In vitro, LPS-induced nitric oxide (NO) production was significantly inhibited by MBF extracts via suppressing the expression of proinflammatory molecules, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-β) and IL-6. Particularly, a dose-dependent inhibition on LPS-induced inflammatory responses was observed following treatment with MBF dichloromethane extract (MBF-DE), in which linoleic acid and ethyl linolenate were identified as two active compounds. Moreover, we elucidated that MBF-DE attenuated LPS-induced inflammatory responses by blocking activation of both NF-κB/p65 and pERK/MAPK pathways. In vivo, DSS-induced acute colitis was significantly ameliorated in MBF-fed mice as gauged by weight loss, colon morphology and histological damage. In addition, MBF-fed MUC2−/− mice displayed significant decrease in intestinal tumor and inflammation incidence compared to control diet-fed group. Overall, our results demonstrated that MBF suppressed the development of intestinal inflammation and tumorgenesis both in vitro and in vivo, and supports the potential of MBF as a therapeutic functional food for testing in human clinical trials.

MicroRNA-223 Attenuates Hypoxia-induced Vascular Remodeling by Targeting RhoB/MLC2 in Pulmonary Arterial Smooth Muscle Cells

There is growing evidence that microRNAs are implicated in pulmonary arterial hypertension (PAH), but underlying mechanisms remain elusive. Here, we identified that miR-223 was significantly downregulated in chronically hypoxic mouse and rat lungs, as well as in pulmonary artery and pulmonary artery smooth muscle cells (PASMC) exposed to hypoxia. Knockdown of miR-223 increased PASMC proliferation. In contrast, miR-223 overexpression abrogated cell proliferation, migration and stress fiber formation. Administering miR-223 agomir in vivo antagonized hypoxia-induced increase in pulmonary artery pressure and distal arteriole muscularization. RhoB, which was increased by hypoxia, was identified as one of the targets of miR-223. Overexpressed miR-223 suppressed RhoB and inhibited the consequent phosphorylation of myosin phosphatase target subunit (MYPT1) and the expression of myosin light chain of myosin II (MLC2), which was identified as another target of miR-223. Furthermore, serum miR-223 levels were decreased in female patients with PAH associated with congenital heart disease. Our study provides the first evidence that miR-223 can regulate PASMC proliferation, migration, and actomyosin reorganization through its novel targets, RhoB and MLC2, resulting in vascular remodeling and the development of PAH. It also highlights miR-223 as a potential circulating biomarker and a small molecule drug for diagnosis and treatment of PAH.

MicroRNA-17-92 regulates myoblast proliferation and differentiation by targeting the ENH1/Id1 signaling axis

Myogenesis is an important biological process that occurs during both skeletal muscle regeneration and postnatal growth. Growing evidence points to the critical role of microRNAs (miRNAs) in myogenesis. Our analysis of miRNA expression patterns reveal that miRNAs of miR-17-92 cluster are dramatically downregulated in C2C12 cells after myogenesis stimulation, are strongly induced in mouse skeletal muscle after injury and decrease steadily thereafter and are downregulated with age in skeletal muscle during mouse and porcine postnatal growth. However, their roles in muscle developmental processes remain elusive. We show that the miR-17-92 cluster promotes mouse myoblast proliferation but inhibits myotube formation. miR-17, -20a and -92a target the actin-associated protein enigma homolog 1 (ENH1). The silencing of ENH1 increased the nuclear accumulation of the inhibitor of differentiation 1 (Id1) and represses myogenic differentiation. Furthermore, the injection of adenovirus expressing miR-20a into the tibialia anterior muscle downregulates ENH1 and delays regeneration. In addition, the downregulation of miR-17-92 during myogenesis is transcriptionally regulated by E2F1. Overall, our results reveal a E2F1/miR-17-92/ENH1/Id1 regulatory axis during myogenesis.