Cellular miR-155 Research Articles

Catalytic hairpin assembled polymeric tetrahedral DNA frameworks for MicroRNA imaging in live cells

Dynamic DNA nanodevices-based assembly is currently well developed for a broad range of analytical applications. However, some problems persist, such as false positives, nuclease digestions, and exclusive interferences with single signal in complex cellular environment. Herein, we have established a method for imaging cellular miR-155, where it induced assembly of two tetrahedral DNA frameworks (TDFs), TDF-1 and TDF-2, both of which had four fluorescence modified hairpins (Cy3 for TDF-1 and Cy5 for TDF-2, respectively) at each angle, into polymeric tetrahedral DNA frameworks (PTDFs). The formation of PTDFs was greatly dependent on miR-155 overexpressed in breast cancer cells since miR-155 drove catalytic hairpin assembly (CHA) reaction by opening the hairpins at the vertices of TDF-1 to hybridize with TDF-2. Upon the completion of hybridization, the miR-155 was released, starting the next cycle of the CHA reaction. Measurements of atomic force microscopy (AFM) and Förster resonance energy transfer (FRET) showed that the formation of PTDFs occurred owing to the multivalent assembly of TDF-1 and TDF-2. By utilizing the formation of PTDFs, miR-155 was detected in a linear range from 0.5nM to 30nM with a 0.35nM limit of determination, enabling the successful imaging of endogenous miR-155 in live cells through the FRET signal from Cy3 to Cy5. These studies demonstrated that this method significantly strengthened the resistance nuclease to digestion and stable ability with exclusive interference.

6mer Seed Toxicity in Viral microRNAs.

SummaryMicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.

A Tiny RNA that Packs a Big Punch: The Critical Role of a Viral miR-155 Ortholog in Lymphomagenesis in Marek's Disease.

MicroRNAs (miRNAs) are small non-coding RNAs that have been identified in animals, plants, and viruses. These small RNAs play important roles in post-transcriptional regulation of various cellular processes, including development, differentiation, and all aspects of cancer biology. Rapid-onset T-cell lymphoma of chickens, namely Marek’s disease (MD), induced by Gallid alphaherpesvirus 2 (GaHV2), could provide an ideal natural animal model for herpesvirus-related cancer research. GaHV2 encodes 26 mature miRNAs derived from 14 precursors assembled in three distinct gene clusters in the viral genome. One of the most highly expressed GaHV2 miRNAs, miR-M4-5p, shows high sequence similarity to the cellular miR-155 and the miR-K12-11 encoded by Kaposi’s sarcoma-associated herpesvirus, particularly in the miRNA “seed region.” As with miR-K12-11, miR-M4-5p shares a common set of host and viral target genes with miR-155, suggesting that they may target the same regulatory cellular networks; however, differences in regulatory function between miR-155 and miR-M4-5p may distinguish non-viral and viral mediated tumorigenesis. In this review, we focus on the functions of miR-M4-5p as the viral ortholog of miR-155 to explore how the virus mimics a host pathway to benefit the viral life cycle and trigger virus-induced tumorigenesis.

Marek’s disease virus type 1 encoded analog of miR-155 promotes proliferation of chicken embryo fibroblast and DF-1 cells by targeting hnRNPAB

Marek’s disease virus type 1 (MDV-1) is a representative oncogenic Alpha herpesvirus that causes an immunosuppressive and neoplastic lymphoproliferative avian disease, namely Marek’s disease (MD). The rapid-onset T-cell lymphoma in chickens induced by MDV-1 has been historically regarded as an ideal natural model for herpesvirus-related cancer research. As a viral analog of cellular miR-155, the MDV-1-encoded miR-M4-5p has been shown to be crucial for the virally-induced MD tumorigenesis. Our previous studies demonstrated that miR-M4-5p induces an over-expression of oncogene c-Myc by targeting LTBP1 and suppressing the TGF-β signaling pathway during MDV-1 infection. We have now further identified the chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) as a new cellular biological target for miR-M4-5p. Suppression of hnRNPAB expression mediated by miR-M4-5p promotes the proliferation, but not the apoptosis, of both primary chicken embryo fibroblasts (CEFs) and transformed chicken fibroblast DF-1 cell line. HnRNPAB is a member of the hnRNP family of proteins that play important roles in normal biological processes as well as cancer development. Our data suggests that the recognition and down-regulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas.

MicroRNA miR-155 Is Necessary for Efficient Gammaherpesvirus Reactivation from Latency, but Not for Establishment of Latency.

MicroRNA-155 (miR-155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper (Tfh) cells. There is in vitro evidence to support a critical role for cellular miR-155 and viral miR-155 homologs in the establishment of gammaherpesvirus latency in B cells. We sought to determine the contribution of miR-155 to the establishment and maintenance of latency in vivo using murine gammaherpesvirus (MHV-68) infection. MHV-68-infected mice deficient in miR-155 exhibited decreases in GC B cells and Tfh cells. However, the frequencies of spleen cells harboring latent MHV-68 genomes were the same in both miR-155-deficient and wild-type (WT) mice. Similar latent loads were also observed in mixed bone marrow chimeric mice, where B cell-extrinsic effects of miR-155 deficiency were normalized. Interestingly, we observed markedly lower efficiency of reactivation from latency in miR-155-deficient cells, indicating an important role for miR-155 in this process. These in vivo data complement previous in vitro studies and lead to the conclusion that miR-155 is not necessary for the establishment or maintenance of gammaherpesvirus latency but that it does affect reactivation efficiency. Gammaherpesvirus infection leads to severe disease in immunosuppressed populations. miR-155 has been shown to play important roles in many pathological processes, including tumorigenesis and diseases caused by an overly aggressive immune response. Our work provides valuable in vivo data showing that miR-155 is dispensable for gammaherpesvirus latency but that it is critical for reactivation from latency, which is a crucial step in the viral life cycle.

Marek׳s disease virus-encoded analog of microRNA-155 activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-β signaling pathway

Marek׳s disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-β binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-β1, with suppression of TGF-β signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

MiRNA modulation of SOCS1 using an influenza A virus delivery system.

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-β cytokines.

MicroRNA-155 and MicroRNA-21 promote the expansion of functional myeloid-derived suppressor cells (IRC5P.473)

Abstract Myeloid-derived suppressor cells (MDSC) are one of the main cell populations that negatively regulate immune responses. However, the mechanism underlying the expansion of MDSC remains unclear. Using miRNA microarray and TaqMan probe-based quantitative RT-PCR assay, we identified microRNA (miR)-155 and miR-21 as the two most upregulated miRNAs during the induction of MDSC from the bone marrow cells by GM-CSF and IL-6. High levels of miR-155 and miR-21 also were detected in bone marrow and spleen MDSC isolated from tumor-bearing mice. Our results also showed that TGF-b promoted the induction of MDSC through upregulating miR-155 and miR-21 expression. Overexpression of miR-155 and miR-21 enhanced whereas depletion of miR-155 and miR-21 reduced the frequencies of cytokine-induced MDSC. Subpopulation analysis indicated that miR-21 and miR-155 induced the expansion of both monocytic and granulocytic MDSC. Furthermore, miR-155 and miR-21 showed a synergistic effect on MDSC induction via targeting SHIP-1 and phosphatase and tensin homolog, respectively, leading to STAT3 activation. Finally, dexamethasone treatment strongly enhanced MDSC expansion through upregulating miR-155 and miR-21 expression, and the effect of dexamethasone on MDSC induction was abolished by depleting cellular miR-155 and miR-21. These results demonstrate a novel miR-155/miR-21-based regulatory mechanism that modulates functional MDSC induction.

MicroRNA-155 and MicroRNA-21 Promote the Expansion of Functional Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSC) are one of the main cell populations that negatively regulate immune responses. However, the mechanism underlying the expansion of MDSC remains unclear. Using miRNA microarray and TaqMan probe-based quantitative RT-PCR assay, we identified microRNA (miR)-155 and miR-21 as the two most upregulated miRNAs during the induction of MDSC from the bone marrow cells by GM-CSF and IL-6. High levels of miR-155 and miR-21 also were detected in bone marrow and spleen MDSC isolated from tumor-bearing mice. Our results also showed that TGF-β promoted the induction of MDSC through upregulating miR-155 and miR-21 expression. Overexpression of miR-155 and miR-21 enhanced whereas depletion of miR-155 and miR-21 reduced the frequencies of cytokine-induced MDSC. Subpopulation analysis indicated that miR-21 and miR-155 induced the expansion of both monocytic and granulocytic MDSC. Furthermore, miR-155 and miR-21 showed a synergistic effect on MDSC induction via targeting SHIP-1 and phosphatase and tensin homolog, respectively, leading to STAT3 activation. Finally, dexamethasone treatment strongly enhanced MDSC expansion through upregulating miR-155 and miR-21 expression, and the effect of dexamethasone on MDSC induction was abolished by depleting cellular miR-155 and miR-21. These results demonstrate a novel miR-155/miR-21-based regulatory mechanism that modulates functional MDSC induction.

Analysis of the mRNA Targetome of MicroRNAs Expressed by Marek’s Disease Virus

ABSTRACTMarek’s disease virus 1 (MDV-1), an oncogenic α-herpesvirus that induces T-cell lymphomas in chickens, serves as model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One MDV-1 miRNA, miR-M4, shares the same “seed” targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155. Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic virus MDV-2. Our analysis identified >1,000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs, and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation. Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation.

A microRNA Encoded by Kaposi Sarcoma-Associated Herpesvirus Promotes B-Cell Expansion In Vivo

The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.

Viral MicroRNA Targetome of KSHV-Infected Primary Effusion Lymphoma Cell Lines

Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and frequently also harbors Epstein-Barr virus (EBV). The expression of KSHV- and EBV-encoded microRNAs (miRNAs) in PELs suggests a role for these miRNAs in latency and lymphomagenesis. Using PAR-CLIP, a technology which allows the direct and transcriptome-wide identification of miRNA targets, we delineate the target sites for all viral and cellular miRNAs expressed in PEL cell lines. The resulting data set revealed that KSHV miRNAs directly target more than 2000 cellular mRNAs, including many involved in pathways relevant to KSHV pathogenesis. Moreover, 58% of these mRNAs are also targeted by EBV miRNAs, via distinct binding sites. In addition to a known viral analog of cellular miR-155, we show that KSHV encodes a viral miRNA that mimics cellular miR-142-3p function. In summary, this study identifies an extensive list of KSHV miRNA targets, which are likely to influence viral replication and pathogenesis.

Critical Role of the Virus-Encoded MicroRNA-155 Ortholog in the Induction of Marek's Disease Lymphomas

Notwithstanding the well-characterised roles of a number of oncogenes in neoplastic transformation, microRNAs (miRNAs) are increasingly implicated in several human cancers. Discovery of miRNAs in several oncogenic herpesviruses such as KSHV has further highlighted the potential of virus-encoded miRNAs to contribute to their oncogenic capabilities. Nevertheless, despite the identification of several possible cancer-related genes as their targets, the direct in vivo role of virus-encoded miRNAs in neoplastic diseases such as those induced by KSHV is difficult to demonstrate in the absence of suitable models. However, excellent natural disease models of rapid-onset Marek's disease (MD) lymphomas in chickens allow examination of the oncogenic potential of virus-encoded miRNAs. Using viruses modified by reverse genetics of the infectious BAC clone of the oncogenic RB-1B strain of MDV, we show that the deletion of the six-miRNA cluster 1 from the viral genome abolished the oncogenicity of the virus. This loss of oncogenicity appeared to be primarily due to the single miRNA within the cluster, miR-M4, the ortholog of cellular miR-155, since its deletion or a 2-nucleotide mutation within its seed region was sufficient to inhibit the induction of lymphomas. The definitive role of this miR-155 ortholog in oncogenicity was further confirmed by the rescue of oncogenic phenotype by revertant viruses that expressed either the miR-M4 or the cellular homolog gga-miR-155. This is the first demonstration of the direct in vivo role of a virus-encoded miRNA in inducing tumors in a natural infection model. Furthermore, the use of viruses deleted in miRNAs as effective vaccines against virulent MDV challenge, enables the prospects of generating genetically defined attenuated vaccines.

Virally Induced Cellular MicroRNA miR-155 Plays a Key Role in B-Cell Immortalization by Epstein-Barr Virus

Infection of resting primary human B cells by Epstein-Barr virus (EBV) results in their transformation into indefinitely proliferating lymphoblastoid cell lines (LCLs). LCL formation serves as a model for lymphomagenesis, and LCLs are phenotypically similar to EBV-positive diffuse large B-cell lymphomas (DLBCLs), which represent a common AIDS-associated malignancy. B-cell infection by EBV induces the expression of several cellular microRNAs (miRNAs), most notably miR-155, which is overexpressed in many tumors and can induce B-cell lymphomas when overexpressed in animals. Here, we demonstrate that miR-155 is the most highly expressed miRNA in LCLs and that the selective inhibition of miR-155 function specifically inhibits the growth of both LCLs and the DLBCL cell line IBL-1. Cells lacking miR-155 are inefficient in progressing through S phase and spontaneously undergo apoptosis. In contrast, three other B-cell lymphoma lines, including two EBV-positive Burkitt's lymphoma cell lines, grew normally in the absence of miR-155 function. These data identify the induction of cellular miR-155 expression by EBV as critical for the growth of both laboratory-generated LCLs and naturally occurring DLBCLs and suggest that targeted inhibition of miR-155 function could represent a novel approach to the treatment of DLBCL in vivo.

A viral microRNA functions as an orthologue of cellular miR-155

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.

Monitoring Serial Change in the Lumen and Outer Wall of Vertebrobasilar Aneurysms

Estimation of the stability of fusiform aneurysms of the basilar artery requires precise monitoring of the luminal and outer wall volumes. In this report we describe the use of MR imaging and 3D postprocessing methods to study the evolution of those aneurysms. Nine patients with fusiform basilar artery aneurysms underwent MR imaging studies covering at least 2 different time points (mean delay between studies, 7.1 +/- 4.6 months). Imaging included multisection 2D T1-weighted fast spin-echo and/or 3D steady-state imaging to assess the outer wall and contrast-enhanced MR angiography to study the lumen. The outer and inner walls were extracted using, respectively, a manual delineation (made by 2 observers) and a thresholding technique. The 2 studies were subsequently coregistered at each time point, as well as between differing time points. Volumes of each vessel component were calculated. Mean volume was 6760 +/- 6620 mm(3) for the outer wall and 2060 +/- 1200 mm(3) for the lumen. Evolution of the lumen and outer wall was highly variable from 1 patient to another, with a trend toward increase of the vessel wall for the largest aneurysms. Interobserver reproducibility for outer wall delineation was on the order of 90%. Combining MR imaging methods to study both the outer wall and lumen with 3D registration tools provides a powerful method for progression of fusiform basilar aneurysmal disease.