Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.     

Elevated miR-155 promotes inflammation in cystic fibrosis by driving hyperexpression of interleukin-8

Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ΔF508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.     

miR-29b regulates migration of human breast cancer cells

microRNAs (miRNAs) are short non-coding RNAs that regulate gene expression by targeting mRNAs, inhibiting the expression of the associated proteins. Although a role for aberrant miRNA expression in cancer has been postulated, the pathophysiologic role and relevance of aberrantly expressed miRNAs in tumor biology has not been established. We evaluated the expression pattern of miRNAs in human breast cancer cells by qPCR, finding out an up-regulated miRNA miR-29b and studying its biological effect by migration assay. We defined a target gene PTEN by bioinformatics approach and western blot. In breast cancer cell line MDA-MB-231 cell, which migrate faster than MCF-7, we observed that miR-29b was highly over-expressed. Inhibition of miR-29b in cultured cells increased the expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, promoting apoptosis, decreasing migration, and decreasing invasion. In contrast, enhanced miR-29b expression by transfection with pre-miR-29b decreased the expression of PTEN and impaired apoptosis, increasing tumor cell migration and invasion. Moreover, PTEN was shown to be a direct target of miR-29b and was also shown to contribute to the miR-29b-mediated effects on cell invasion. Modulation of miR-29b altered the role of PTEN involved in cell migration and invasion. Aberrant expression of miR-29b, which modulates PTEN expression, can contribute to migration, invasion, and anti-apoptosis.     

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3'-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5'-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5'-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5'-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3' region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122-HCV interaction, and the broader consequences of miRNAs binding to 5'-UTR sites.     

MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.     

Regulation and biological function of the liver-specific miR-122

miRNAs (microRNAs) are important regulators of gene expression. In higher eukaryotes, the tightly controlled expression of different miRNAs, each of which regulates multiple target mRNAs, is crucial for the maintenance of tissue type and the control of differentiation. miR-122 is a highly liver-specific miRNA that is important in hepatitis C virus infection, cholesterol metabolism and hepatocellular carcinoma. In the present review, we discuss the effects of miR-122 on liver physiology and pathology. Recent evidence of pathways involved in the regulation of miR-122 expression is also considered.