Gld2 activity is regulated by phosphorylation in the N-terminal domain

The de-regulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. Human miR-122 is required for Hepatitis C proliferation, and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyses the post-transcriptional addition of a single adenine residue (A + 1) to the 3ʹ-end of miR-122, enhancing its stability. Gld2 activity is inhibited by binding to the Hepatitis C virus core protein during HepC infection, but no other mechanisms of Gld2 regulation are known. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity, revealing tumour suppressor miRNAs as a previously unknown target of Akt1-dependent signalling

MicroRNA-122 in patients with hepatitis B and hepatitis B virus-associated hepatocellular carcinoma

Hepatitis B virus (HBV) infection is known as a serious problem in the domain of public health and approximately 350 million people across the world are affected with this infectious disease. As well, microRNAs are recognized as a type of small non-coding RNAs that can be widely used as a diagnostic biomarker and prognosis method of special diseases. In this respect, microRNA-122 or miR-122 can play a significant role in the pathogenesis of several hepatic diseases. Given the importance of microRNA-122 in the liver as well as its pathology, this study focused on the potential functions of microRNA-122 in pathogenesis, diagnosis, and treatment of HBV infection. In this regard, the findings of previous studies had indicated that expression of microRNA-122 in patients with HBV infection could be significantly deregulated. The results of this study were consistent with the idea that diagnosis and treatment of this infectious disease using microRNA-122 could be an efficient method. Keywords Author Keywords:Hepatitis B virus (HBV); microRNA-122; hepatocellular carcinoma (HCC); biomarker KeyWords Plus:REGION CONFERS RISK; MIRNA-122-BINDING SITE; CIRCULATING MICRORNAS; REGULATORY CIRCUITRY; CELL-PROLIFERATION; VIRAL REPLICATION; DOWN-REGULATION; LIVER-CANCER; MIR-122; EXPRESSIO

The Regulatory Role of MicroRNA in Hepatitis-B Virus-Associated Hepatocellular Carcinoma (HBV-HCC) Pathogenesis.

The incidence and mortality of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HBV-HCC) is an intractable public health problem in developing countries that is compounded by limited early detection and therapeutic options. Despite the early promise of utilizing the regulatory role of miRNA in liver cancer, this field remains largely in the work-in-progress phase. This exploratory review paper adopts a broad focus in order to collate evidence of the regulatory role of miRNA in each stage of the HBV-HCC continuum. This includes the regulatory role of miRNA in early HBV infection, chronic inflammation, fibrosis/cirrhosis, and the onset of HCC. The paper specifically investigates HBV dysregulated miRNA that influence the expression of the host/HBV genome in HBV-HCC pathogenesis and fully acknowledges that this does not cover the full spectrum of dysregulated miRNA. The sheer number of dysregulated miRNA in each phase support a hypothesis that future therapeutic interventions will need to consider incorporating multiple miRNA panels

Global analysis of HBV-mediated changes to the primary hepatocyte transcriptome and metabolome

Chronic infection with hepatitis B virus (HBV) remains a significant health concern, with between 350-500 million people chronically infected worldwide. Approximately 25% of chronically infected individuals will go on to develop HBVassociated hepatocellular carcinoma (HCC), making chronic infection with HBV the leading risk factor for developing HCC. With the high incidence and mortality of HCC, it is important to fully understand the mechanisms that lead to the development of HBV-associated HCC. HBV requires a complex network of hostvirus interactions to meet its requirements for successful replication. Each of these host-virus interactions, over a decades-long chronic infection, could significantly alter the physiology of an infected hepatocyte, the target of HBV infection, and ultimately contribute to the oncogenic potential of HBV. Typically, studies of these host-virus interactions have focused on a single factor or pathway to characterize contributions to HBV replication or HBV-associated disease, but these studies have generally not considered hepatocyte physiology as a whole. We hypothesized that using broad, transcriptomic- and metabolomic -based technologies would allow us to establish a better understanding of the complexity of the host-virus interaction mediated by HBV and how an HBV infection affects overall hepatocyte physiology. To achieve this, we utilized an ex-vivo primary rat hepatocyte model and defined transcriptome-wide, HBV-mediated changes to gene expression. We also utilized metabolomic profiling to assess the impact of HBV, and the HBV X protein (HBx), on overall hepatocyte metabolism and correlated these changes to HBV-mediated changes in gene expression. Using this approach, we identified significant alterations of many genes and pathways central to hepatocyte physiology, including cell cycle regulation, lipid metabolism, and energy metabolism. Our results simultaneously identified multiple HBV-mediated changes to hepatocyte physiology, increasing our understanding of the complex relationship between HBV and an infected hepatocyte. Additionally, the identification of HBV-regulated genes will serve as the basis for future studies in understanding the physiological impact of an HBV infection. Together, these findings will allow a better understanding of HBV-mediated affects on hepatocyte physiology that could ultimately contribute to the development of HBV-associated disease, potentially guiding the generation of novel therapeutics and strategies to prevent HBV-associated HCC.Ph.D., Microbiology and Immunology -- Drexel University, 201

A Genomic Portrait of Hepatitis C Virus and MicroRNA-122

Hepatitis C virus (HCV) uniquely requires the liver specific microRNA-122 (miR- 122) for replication, yet global effects on endogenous microRNA (miRNA) targets during infection are unexplored. In this body of work, we employed highthroughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection. We demonstrate robust AGO binding on the 5\u27 untranslated region of HCV RNA at known and predicted miR-122 sites, thereby establishing conclusive biochemical evidence of endogenous miR-122 action on HCV RNA that firmly agrees with previous genetic evidence. We further characterize novel AGO binding on HCV RNA to determine its dependence on miR-122, miRNAs generally, replication competence and time. These results establish an unbiased interaction landscape between HCV RNA and cellular miRNAs, mostly miR-122. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 sponge effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. Based on these results, we describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets. This in turn may provide an environment fertile for the long-term oncogenic potential of HCV. This last point presented a fitting entree into miR-122 biology, given its known tumor suppressive activity in the liver. To conclude this work, we performed AGO-CLIP in miR-122 knockout mouse livers as well as in human liver samples, to determine the in vivo targetome for this miRNA across two species. Surprisingly, we discovered widespread and non-canonical miR-122 binding throughout the transcriptome. Furthermore, a substantial fraction of this binding was not conserved between mouse and human transcriptomes, despite the fact that miR-122 is highly conserved. These results, in concert with AGOCLIP in HCV infected cells, point to a model where HCV may have evolved the use of miR-122 for its high abundance and its well buffered capacity to be inhibited with minimal detrimental effects to the host, and perhaps benefits for the virus. In sum, this thesis reveals how miR-122 is redistributed in the cell following HCV infection. As a molecular mechanism, chronic inhibition of miR-122 by HCV RNA is proposed to impact, and may very well help induce, the complex constellation of liver diseases that characterize this infection in humans

Syntheses and applications of small molecule inhibitors of miRNAs miR-21 and miR-122

MicroRNAs (miRNAs) are regulatory RNA molecules of 22 nucleotides that (in part) control up to 60% of all genes in humans. They act by binding to the 3' untranslated regions of target messenger RNAs, leading either to translational repression or mRNA degradation. In addition to being involved in the regulation of several fundamental cellular processes, the misregulation of miRNAs has been linked to a wide range of diseases including cancer. Particularly, miR-21 is significantly upregulated in nearly all types of human cancers, and its overexpression is often associated with poor prognosis. The downregulation of miR-122 is found in more than 70% of hepatocellular carcinoma cases and miR-122 is a required factor for the replication of the HCV virus. The modulation of miRNA function is commonly achieved using oligonucleotide agents. However, compared to oligonucleotides, small molecules have several advantages, such as fast activity, systemic delivery, and excellent cell permeability. Taking advantage of luciferase-based reporters, two separate high-throughput screens of >300,000 compounds each, were conducted to discover new small molecule inhibitors of miR-21 or miR-122. Several hit compounds were re-synthesized, their ability to inhibit miR-21 was validated, and the most promising compounds were investigated by SAR studies, which revealed two additional, structurally diverse classes of miR-21 inhibitors. Similarly, extensive SAR studies of previously discovered miR-122 inhibitors were performed in order to better understand the molecular requirements for the miR-122 inhibitory activity. The hit compounds identified in the HTS were analyzed through secondary assays that led to the identification of two new promising miR-122 inhibitors. Furthermore, the knowledge gained during the SAR studies was further used to synthesize several small molecule miR-21/miR-122 inhibitors as probes to explore their mechanisms of action. MicroRNAs represent promising, novel drug targets, and small molecule miRNA inhibitors provide tools to study the molecular mechanisms of miRNA biogenesis and have the potential to be new therapeutic agents for the treatment of cancers and viral infections