Deming Gou

MicroRNA: past and present.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) non-coding RNAs that participate in gene regulation. MiRNAs confer their regulation at a post-transcriptional level, where they either cleave or repress translation of mRNAs. Over 3000 identified mature miRNAs exist in species ranging from plants to humans, suggesting that they are ancient players in gene regulation. A relatively small number of miRNAs have been experimentally tested for their function. Of those tested, functions including cell differentiation, proliferation, apoptosis, anti-viral defense and cancer have been proposed. Improved software programs are now able to predict the targets of miRNAs in a more efficient manner, thus facilitating the elucidation of miRNA function. Furthermore, methods such as real-time PCR and microarray have been enhanced for studying miRNA expression. Using these tools, scientists are able to discover novel functions for miRNAs. It is possible that miRNAs will be revealed as having a role in virtually every aspect of gene regulation. This review guides readers through the biogenesis of miRNAs, their mechanism of action on their target mRNAs, the functional outcomes of their action on mRNAs and the current techniques to investigate these processes.

MicroRNA-127 modulates fetal lung development

MicroRNAs (miRNAs) are small endogenous RNAs and are widely regarded as one of the most important regulators of gene expression in both plants and animals. To define the roles of miRNAs in fetal lung development, we profiled the miRNA expression pattern during lung development with a miRNA microarray. We identified 21 miRNAs that showed significant changes in expression during lung development. These miRNAs were grouped into four distinct clusters based on their expression pattern. Cluster 1 contained miRNAs whose expression increased as development progressed, while clusters 2 and 3 showed the opposite trend of expression. miRNAs in cluster 4 including miRNA-127 (miR-127) had the highest expression at the late stage of fetal lung development. Quantitative real-time PCR validated the microarray results of six selected miRNAs. In situ hybridization demonstrated that miR-127 expression gradually shifted from mesenchymal cells to epithelial cells as development progressed. Overexpression of miR-127 in fetal lung organ culture significantly decreased the terminal bud count, increased terminal and internal bud sizes, and caused unevenness in bud sizes, indicating improper development. These findings suggest that miR-127 may have an important role in fetal lung development.

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.

Identification of rat lung-specific microRNAs by microRNA microarray: valuable discoveries for the facilitation of lung research

BackgroundAn important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer.ResultsIn this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis.ConclusionThe identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Trans-differentiation of Alveolar Epithelial Type II Cells to Type I Cells Involves Autocrine Signaling by Transforming Growth Factor β1 through the Smad Pathway

Type II alveolar epithelial cells (AEC II) proliferate and transdifferentiate into type I alveolar epithelial cells (AEC I) when the normal AEC I population is damaged in the lung alveoli. We hypothesized that signaling by transforming growth factor β1 (TGF β1), through its downstream Smad proteins, is involved in keeping AEC II quiescent in normal cells and its altered signaling may be involved in the trans-differentiation of AEC II to AEC I. In the normal lung, TGF β1 and Smad4 were highly expressed in AEC II. Using an in vitro cell culture model, we demonstrated that the trans-differentiation of AEC II into AEC I-like cells began with a proliferative phase, followed by a differentiation phase. The expression of TGF β1, Smad2, and Samd3 and their phosphorylated protein forms, and cell cycle inhibitors, p15Ink4b and p21Cip1, was lower during the proliferative phase but higher during the differentiation phase. Furthermore, cyclin-dependent kinases 2, 4, and 6 showed an opposite trend of expression. TGF β1 secretion into the media increased during the differentiation phase, indicating an autocrine regulation. The addition of TGF β1 neutralizing antibody after the proliferative phase and silencing of Smad4 by RNA interference inhibited the trans-differentiation process. In summary, our results suggest that the trans-differentiation of AEC II to AEC I is modulated by signaling through the Smad-dependent TGF β1 pathway by altering the expression of proteins that control the G1 to S phase entry in the cell cycle.

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.

Gene silencing in mammalian cells by PCR-based short hairpin RNA

RNA interference (RNAi) provides a powerful tool to silence genes in a sequence‐specific manner in a variety of systems. However, not all sequences are effective in the RNAi‐mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)‐based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two‐step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and β‐galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning.

Trans-differentiation of alveolar epithelial type II cells to type I cells involves autocrine signaling by TGF β1 through the Smad pathway

Type II alveolar epithelial cells (AEC II) proliferate and transdifferentiate into type I alveolar epithelial cells (AEC I) when the normal AEC I population is damaged in the lung alveoli. We hypothesized that signaling by transforming growth factor β1 (TGF β1), through its downstream Smad proteins, is involved in keeping AEC II quiescent in normal cells and its altered signaling may be involved in the trans-differentiation of AEC II to AEC I. In the normal lung, TGF β1 and Smad4 were highly expressed in AEC II. Using an in vitro cell culture model, we demonstrated that the trans-differentiation of AEC II into AEC I-like cells began with a proliferative phase, followed by a differentiation phase. The expression of TGF β1, Smad2, and Samd3 and their phosphorylated protein forms, and cell cycle inhibitors, p15Ink4b and p21Cip1, was lower during the proliferative phase but higher during the differentiation phase. Furthermore, cyclin-dependent kinases 2, 4, and 6 showed an opposite trend of expression. TGF β1 secretion into the media increased during the differentiation phase, indicating an autocrine regulation. The addition of TGF β1 neutralizing antibody after the proliferative phase and silencing of Smad4 by RNA interference inhibited the trans-differentiation process. In summary, our results suggest that the trans-differentiation of AEC II to AEC I is modulated by signaling through the Smad-dependent TGF β1 pathway by altering the expression of proteins that control the G1 to S phase entry in the cell cycle.

A novel approach for the construction of multiple shRNA expression vectors.

The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence‐specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time‐consuming. Here, we describe a simple, quick and low‐cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies. Copyright

A novel function of ionotropic γ-aminobutyric acid receptors involving alveolar fluid homeostasis

Polarized distribution of chloride channels on the plasma membrane of epithelial cells is required for fluid transport across the epithelium of fluid-transporting organs. Ionotropic γ-aminobutyric acid receptors are primary ligand-gated chloride channels that mediate inhibitory neurotransmission. Traditionally, these receptors are not considered to be contributors to fluid transport. Here, we report a novel function of γ-aminobutyric acid receptors involving alveolar fluid homeostasis in adult lungs. We demonstrated the expression of functional ionotropic γ-aminobutyric acid receptors on the apical plasma membrane of alveolar epithelial type II cells. γ-Aminobutyric acid significantly increased chloride efflux in the isolated type II cells and inhibited apical to basolateral chloride transport on type II cell monolayers. Reduction of the γ-aminobutyric acid receptor π subunit using RNA interference abolished the γ-aminobutyric acid-mediated chloride transport. In intact rat lungs, γ-aminobutyric acid inhibited both basal and β agonist-stimulated alveolar fluid clearance. Thus, we provide molecular and pharmacological evidence that ionotropic γ-aminobutyric acid receptors contribute to fluid transport in the lung via luminal secretion of chloride. This finding may have the potential to develop clinical approaches for pulmonary diseases involving abnormal fluid dynamics.