Ahmed Ihab Abdelaziz

Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

ABSTRACT Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLNlow subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLNlow cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.

Development of a Second Version of the Cobas AmpliPrep/Cobas TaqMan Hepatitis C Virus Quantitative Test with Improved Genotype Inclusivity

ABSTRACT Hepatitis C virus (HCV) RNA measurement has been facilitated by the introduction of real-time PCR-based assays with low limits of detection and broad dynamic ranges for quantification. In the present study, the performance of two second-version prototypes of the Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test (CAP/CTM v2) with decreased sample input volume and improved genotype inclusivity was investigated. A total of 232 serum and plasma samples derived from patients with chronic hepatitis C (genotype 1 [GT1], n = 108; GT2, n = 8; GT3, n = 24; GT4, n = 87; GT5, n = 3; and GT6, n = 2) were processed in parallel with the Cobas AmpliPrep/Cobas TaqMan HCV Test (CAP/CTM), Cobas Amplicor HCV Monitor Test v2.0 (CAM), and two second-version prototype formulations of CAP/CTM, Mastermix 1 (MMx1) and MMx2. In addition, three GT4 transcripts containing rare variant sequences were tested. The mean log10 HCV RNA differences for the best-performing CAP/CTM v2/MMx2 formulation in comparison to CAM were −0.05, 0.05, −0.12, −0.10, −0.44, and −0.29 for patients with GT1, GT2, GT3, GT4, GT5, and GT6 infections, respectively. GT1, GT2, and GT4 samples including isolates with known variants within the 5′ untranslated region (G145A, A165T) that were underquantified with CAP/CTM were correctly quantified with the second-version prototype. In addition, CAP/CTM v2 was able to accurately quantify the three transcripts with rare variant sequences. In conclusion, CAP/CTM v2 accurately quantifies HCV RNA across all HCV genotypes, including specimens with rare polymorphisms previously associated with underquantification.

Correcting the expression of miRNA-155 represses PP2Ac and enhances the release of IL-2 in PBMCs of juvenile SLE patients

MicroRNA-155 is involved in immune cell, differentiation, maturation and function. MiR-155 showed variable dysregulated expression in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients. MiR-155 was previously confirmed to directly target CAMP response element binding protein (CREB), which was previously identified as a positive regulator of protein phosphatase 2A (PP2A). PP2A is a key negative regulator of interleukin-2, which is an important immune modulator and was previously shown to be decreased in SLE. In this study we aimed at investigating the regulation of PP2A by miR-155 and hence its role in juvenile SLE disease pathogenesis. MiR-155 showed significant downregulation in PBMCs from juvenile SLE and juvenile familial Mediterranean fever (FMF) and significant upregulation in PBMCs from juvenile idiopathic arthritis (JIA) patients. In SLE, miR-155 expression was negatively correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score and proteinuria and was positively correlated with white blood cell (WBC) count. The mRNA of the catalytic subunit of PP2A (PP2Ac) showed significant upregulation in PBMCs from SLE and FMF but not in JIA patients. Additionally, the relative expression of PP2Ac mRNA was positively correlated with SLEDAI score. Forced expression of miR-155 led to decreased relative expression of PP2Ac mRNA and increased IL-2 release in cultured-stimulated PBMCs. This study suggests for the first time the possible role of an miR-155-PP2Ac loop in regulating IL-2 release and identifies miR-155 as a potential therapeutic target in juvenile SLE disease through relieving IL-2 from the inhibitory role of PP2A.

miR‐615‐5p is restrictedly expressed in cirrhotic and cancerous liver tissues and its overexpression alleviates the tumorigenic effects in hepatocellular carcinoma

microRNAs aberrant behavior in heptocellular carcinoma (HCC) plays a major role in HCC pathogenesis. miR‐615‐5p expression has never been evaluated in HCC. We showed that miR‐615‐5p was preferentially expressed in HCC, cirrhotic liver tissues and HCC cell lines, but undetected in normal livers. Forced miR‐615‐5p expression in HCC cell lines led to significant decrease in cell growth and migration. In‐silico predication revealed insulin‐like growth factor‐II (IGF‐II) as a potential downstream target for miR‐615‐5p. Forcing the expression of miR‐615‐5p showed downregulation of IGF‐II mRNA, as well as inhibition of the luciferase activity in a luciferase reporter vector harboring the IGF‐II‐3′UTR target sequence. miR‐615‐5p acts as tumor‐suppressor in HCC through targeting IGF‐II.

Repressed induction of interferon-related microRNAs miR-146a and miR-155 in peripheral blood mononuclear cells infected with HCV genotype 4

MicroRNAs regulate the expression of many genes and subsequently control various cellular processes, such as the immune response to viral infections mediated by type I interferon (IFN). In this study, the expression pattern of two interferon‐related microRNAs, miR‐146a and miR‐155, was examined in healthy and HCV‐genotype‐4‐infected peripheral blood mononuclear cells (PBMCs) using qRT‐PCR. In contrast to other viral infections, the expression pattern was similar in both healthy and infected PBMCs. This could be attributed to attenuation of IFN pathway by HCV, which was assessed by investigating the expression of MxA, an interferon‐stimulated gene, that showed lower expression in HCV‐infected PBMCs. To determine the site of interference of HCV in the IFN pathway, expression of both microRNAs was examined following stimulation of PBMCs with IFN‐α2a, an activator of the JAK/STAT pathway as well as with imiquimod, a toll‐like receptor‐7 (TLR‐7) agonist that promotes interferon release. IFN stimulation induced the expression of miR‐146a and miR‐155 in HCV‐infected and healthy PBMCs. Stimulation with imiquimod led to a down‐regulation of both microRNAs in infected PBMCs, while it increased their expression in healthy PBMCs, indicating that HCV might interfere with miR‐146a and miR‐155 expression at sites upstream of interferon release, specifically in the TLR‐7 pathway. The pattern of expression of both miR‐146a and miR‐155 was very similar with a strong positive correlation, but showed no correlation to the patients’ clinical or histopathological parameters or response to treatment. In conclusion, HCV infection might repress the induction of miR‐146a and miR‐155 by interfering with TLR‐7 signaling.

MicroRNA-181a - a tale of discrepancies.

MicroRNAs (miRNAs) are short noncoding RNAs that act as post-transcriptional regulators. The low complementarity required between the sequences of a miRNA and its target mRNA enables a single miRNA to act on a large range of targets. Thus miRNAs have an intersecting complex effect that spans a multiplicity of pathways and processes. In this review, the different roles of a vital miRNA, miR-181a, in physiological and pathological developments are collated in an attempt to highlight the intersections of such processes and to show how the deregulation of miR-181a could in one context drive malignancy, whereas in another it can lead to autoimmunity. Such deregulation could be related to the faulty levels of one of its own targets, p53, which was recently reported to control an array of miRNAs, one of which is miR-181a. This sheds light on a hidden loop of chaos behind chronic diseases such as autoimmunity and cancer.

Hepatocytes that express variants of cyclophilin A are resistant to HCV infection and replication.

BACKGROUND & AIMS Hepatitis C virus (HCV) uses several host factors to infect and replicate in human hepatocytes. Cyclophilin A (CypA) is required for viral replication, and CypA inhibitors are in development. We investigated the effects of nonsynonymous single nucleotide polymorphisms (SNPs) in the region of peptidyl-prolyl isomerase A (PPIA) that encodes CypA on HCV infection and replication of human hepatocytes. METHODS We used a combination of virologic, biochemical, and genetic approaches to investigate the effects of PPIA variants on HCV replication in cultured Huh-7.5 cells. We reduced levels of CypA in these cells using small hairpin RNAs (shRNAs). RESULTS Using shRNAs, we showed that CypA was required for replication of HCV in Huh-7.5 cells and identified 3 SNPs in PPIA that protected cells from HCV entry or replication. Levels of HCV RNA were reduced 3-4 log in cells homozygous for the variant alleles; release of new particles was also reduced, but viral entry was not affected. The effects of the variant alleles were recessive and stronger for preventing replication of full-length HCV genomes than subgenomes. CypA inhibitors prevented replication of residual HCV in hepatocytes. The variants appeared to destabilize the CypA protein; the single amino acid changes led to rapid degradation of the protein. CONCLUSIONS We identified variants in PPIA that destabilize its product, CypA, and prevent HCV infection and replication. These findings indicate mechanisms by which some cells might be resistant to HCV infection and that CypA is a good therapeutic target.

miR-1275: A single microRNA that targets the three IGF2-mRNA-binding proteins hindering tumor growth in hepatocellular carcinoma

This study aimed to identify a single miRNA or miR (microRNA) which regulates the three insulin‐like growth factor‐2‐mRNA‐binding proteins (IGF2BP1, 2 and 3). Bioinformatics predicted miR‐1275 to simultaneously target the three IGF2BPs, and screening revealed miR‐1275 to be underexpressed in hepatocellular carcinoma (HCC) tissues. Transfection of HuH‐7 cells with miR‐1275 suppressed IGF2BPs expression and all three IGF2BPs were confirmed as targets of miR‐1275. Ectopic expression of miR‐1275 and knockdown of IGF2BPs inhibited malignant cell behaviors, and also reduced IGF1R protein and mRNA. Finally IGF1R was validated as a direct target of miR‐1275. These findings indicate that the tumor‐suppressor miR‐1275 can control HCC tumor growth partially through simultaneously regulating the oncogenic IGF2BPs and IGF1R.

Expression of insulin-like growth factor-II, matrix metalloproteinases, and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients

Background/aim: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC. Patients/methods: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR. Results: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P = 0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value = 0.0138. Conclusion: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.

Repression of miR-17-5p with elevated expression of E2F-1 and c-MYC in non-metastatic hepatocellular carcinoma and enhancement of cell growth upon reversing this expression pattern

E2F-1, c-MYC, and miR-17-5p is a triad of two regulatory loops: a negative and a positive loop, where c-MYC induces the expression of E2F-1 that induces the expression of miR-17-5p which in turn reverses the expression of E2F-1 to close the loop. In this study, we investigated this triad for the first time in hepatocellular carcinoma (HCC), where miR-17-5p showed a significant down-regulation in 23 non-metastatic HCC biopsies compared to 10 healthy tissues; however, E2F-1 and c-MYC transcripts were markedly elevated. Forced over-expression of miR-17-5p in HuH-7 cells resulted in enhanced cell proliferation, growth, migration and clonogenicity with concomitant inhibition of E2F-1 and c-MYC transcripts expressions, while antagomirs of miR-17-5p reversed these events. In conclusion, this study revealed a unique pattern of expression for miR-17-5p in non-metastatic HCC patients in contrast to metastatic HCC patients. In addition we show that miR-17-5p is the key player among the triad that tumor growth and spread.