miR-198 Inhibits HIV-1 Gene Expression and Replication in Monocytes and Its Mechanism of Action Appears To Involve Repression of Cyclin T1

Cyclin T1 is a regulatory subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is also required for Tat transactivation of HIV-1 LTR-directed gene expression. Translation of Cyclin T1 mRNA has been shown to be repressed in human monocytes, and this repression is relieved when cells differentiate to macrophages. We identified miR-198 as a microRNA (miRNA) that is strongly down-regulated when monocytes are induced to differentiate. Ectopic expression of miR-198 in tissue culture cells reduced Cyclin T1 protein expression, and plasmid reporter assays verified miR-198 target sequences in the 3′ untranslated region (3′UTR) of Cyclin T1 mRNA. Cyclin T1 protein levels increased when an inhibitor of miR-198 was transfected into primary monocytes, and overexpression of miR-198 in primary monocytes repressed the normal up-regulation of Cyclin T1 during differentiation. Expression of an HIV-1 proviral plasmid and HIV-1 replication were repressed in a monocytic cell line upon overexpression of miR-198. Our data indicate that miR-198 functions to restrict HIV-1 replication in monocytes, and its mechanism of action appears to involve repression of Cyclin T1 expression

Serum MicroRNA-21 as Marker for Necroinflammation in Hepatitis C Patients with and without Hepatocellular Carcinoma

Background: MicroRNA-21 (miR-21) is up-regulated in tumor tissue of patients with malignant diseases, including hepatocellular carcinoma (HCC). Elevated concentrations of miR-21 have also been found in sera or plasma from patients with malignancies, rendering it an interesting candidate as serum/plasma marker for malignancies. Here we correlated serum miR-21 levels with clinical parameters in patients with different stages of chronic hepatitis C virus infection (CHC) and CHC-associated HCC. Methodology/Principal Findings: 62 CHC patients, 29 patients with CHC and HCC and 19 healthy controls were prospectively enrolled. RNA was extracted from the sera and miR-21 as well as miR-16 levels were analyzed by quantitative real-time PCR; miR-21 levels (normalized by miR-16) were correlated with standard liver parameters, histological grading and staging of CHC. The data show that serum levels of miR-21 were elevated in patients with CHC compared to healthy controls (P<0.001); there was no difference between serum miR-21 in patients with CHC and CHC-associated HCC. Serum miR-21 levels correlated with histological activity index (HAI) in the liver (r = −0.494, P = 0.00002), alanine aminotransferase (ALT) (r = −0.309, P = 0.007), aspartate aminotransferase (r = −0.495, P = 0.000007), bilirubin (r = −0.362, P = 0.002), international normalized ratio (r = −0.338, P = 0.034) and γ-glutamyltransferase (r = −0.244, P = 0.034). Multivariate analysis revealed that ALT and miR-21 serum levels were independently associated with HAI. At a cut-off dCT of 1.96, miR-21 discriminated between minimal and mild-severe necroinflammation (AUC = 0.758) with a sensitivity of 53.3% and a specificity of 95.2%. Conclusions/Significance: The serum miR-21 level is a marker for necroinflammatory activity, but does not differ between patients with HCV and HCV-induced HCC

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Hepatitis C Virus Infection and Hepatic Stellate Cell Activation Downregulate miR-29: miR-29 Overexpression Reduces Hepatitis C Viral Abundance in Culture

Background. Chronic hepatitis C virus (HCV)–induced liver fibrosis involves upregulation of transforming growth factor (TGF)–β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. Methods. TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. Results. This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. Conclusions. HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.University of Iowa. College of MedicineUniversity of Iowa. College of Medicine (Translational Pilot Grant)University of Iowa. Levitt Center for Viral Pathogenesis (Translational Pilot Grant)University of Iowa (Dean's Fellowship)National Institute of Allergy and Infectious Diseases (U.S.) (Predoctoral Training Grant (T32AI007533))United States. Veterans Administration (Merit Review Grant)National Institutes of Health (U.S.) (Grant R21 DK068453-01A1)University of Iowa (Carver Trust Foundation)United States. Dept. of Energy (Computational Sciences Graduate Fellowship