miR-151a enhances Slug dependent angiogenesis.

MicroRNAs (miRs) are small non-coding RNAs, that modulate cognate gene expression either by inducing mRNA degradation or by blocking translation, and play crucial and complex roles in tissue homeostasis and during disease initiation and progression. The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis and aberrant angiogenesis is associated with pathological conditions such as ischemia and cancer. We have previously established that miR-151a functions as an onco-miR in non-small cell lung cancer (NSCLC) cells by inducing partial EMT and enhancing tumor growth. Here, we identify anti-miR-151a as a molecule that promotes endothelial cell contacts and barrier properties, suggesting that miR-151a regulates cell-cell junctions. We find that induced miR-151a expression enhances endothelial cell motility and angiogenesis and these functions depend on miR-151a-induced Slug levels. Moreover, we show that miR-151a overexpression enhances tumor-associated angiogenesis in 3D vascularized tumor spheroid assays. Finally, we verify that miR-151a is expressed in the vasculature of normal lung and NSCLC tissue. Our results suggest that miR-151a plays multi-faceted roles in the lung, by regulating multiple functions (cell growth, motility, partial EMT and angiogenesis) in distinct cell types.

Interferon-Inducible MicroRNA miR-128 Modulates HIV-1 Replication by Targeting TNPO3 mRNA.

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR), miR-128, represses retrotransposon long interspaced element 1 (L1) by a dual mechanism, namely, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins TNPO1, TNPO2, and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication of TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that type I interferon (IFN)-inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly downregulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wild-type (WT), replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by overexpression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not murine leukemia virus (MLV) infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in antiviral defense in human cells, namely inhibiting HIV-1 replication by altering the cellular milieu through targeting factors that include TNPO3.IMPORTANCE HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced, and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study, we identified the interferon-inducible microRNA (miR) miR-128, a novel antiviral mediator that suppresses the expression of the host gene TNPO3, which is known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.

miR-128 inhibits telomerase activity by targeting TERT mRNA.

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes.

Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.

SAMHD1 is a phosphohydrolase maintaining cellular dNTP homeostasis but also acts as a critical regulator in innate immune responses due to its antiviral activity and association with autoimmune disease, leading to aberrant activation of interferon. SAMHD1 expression is differentially regulated by interferon in certain primary cells, but the underlying mechanism is not understood. Here, we report a detailed characterization of the promotor region, the 5'- and 3'-untranslated region (UTR) of SAMHD1, and the mechanism responsible for the cell type-dependent up-regulation of SAMHD1 protein by interferon. We demonstrate that induction of SAMHD1 by type I and II interferons depends on 3'-UTR post-transcriptional regulation, whereas the promoter drives basal expression levels. We reveal novel functional target sites for the microRNAs miR-181a, miR-30a, and miR-155 in the SAMHD1 3'-UTR. Furthermore, we demonstrate that down-regulation of endogenous miR-181a and miR-30a levels inversely correlates with SAMHD1 protein up-regulation upon type I and II interferon stimulation in primary human monocytes. These miRNAs are not modulated by interferon in macrophages or dendritic cells, and consequently protein levels of SAMHD1 remain unchanged. These results suggest that SAMHD1 is a non-classical interferon-stimulated gene regulated through cell type-dependent down-regulation of miR-181a and miR-30a in innate sentinel cells.

Transposable elements and miRNA: Regulation of genomic stability and plasticity.

Transposable elements, the class of mobile DNA sequences that change their copies or positions within the genome have an ever increasing role in shaping the genetic and evolutionary landscape. Approximately half of the mammalian genome is composed of repetitive elements, including LINE-1 (L1) elements. Because of their ability to "copy and paste" into other regions of the genome, their activation represent an opportunity as well as a threat, as L1-induced mutations results in genomic instability and plasticity. On one hand L1 retrotransposition and integration fosters genomic diversity and on the other, de-repressed L1 functions as a driver of diseases such as cancer. The regulation of L1 is an area of intense research and novel epigenetic mechanisms have recently been discovered to now include DNA methylation, histone modifications, and miR-induced L1 silencing. During development, reprogramming and in transformed cells, specific classes of repetitive elements are upregulated, presumably due to the loss of epigenetic regulation in this process, increasing the risk of L1-induced mutations. Here we discuss how miR regulation of L1 activation fits into the complex picture of L1 repression in somatic cells and touch on some of the possible implications.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism.

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.