The Role of the MYC/miR-150/MYB/ZDHHC11 Network in Hodgkin Lymphoma and Diffuse Large B-Cell Lymphoma

We previously described involvement of the MYC/miR-150/MYB/ZDHHC11 network in the growth of Burkitt lymphoma (BL) cells. Here we studied the relevance of this network in the two other B-cell lymphomas: Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL). Expression levels of the network components were assessed at the RNA and protein level. The effect of modulating levels of the network components on cell growth was determined through GFP competition assay. AGO2-RNA immunoprecipitation was performed to validate targeting by miR-150. Expression levels of MYC, MYB and ZDHHC11 were increased, while miR-150 levels were decreased similar to the pattern observed in BL. The knockdown of MYC, MYB and ZDHHC11 decreased the growth of HL and DLBCL cells. In contrast, overexpression of miR-150 did not induce clear phenotypes in HL, and limited the effects in DLBCL. This could not be explained by the differences in overexpression levels. Furthermore, we showed that in HL, ZDHHC11 and MYB are efficiently targeted by miR-150. To conclude, MYC, MYB and ZDHHC11 are critical for the growth of HL and DLBCL cells consistent with the role observed in BL cells, while low endogenous miR-150 levels appeared to be less critical for the growth of HL and DLBCL cells despite the effective targeting of ZDHHC11 and MYB

The miR-26b-5p/KPNA2 Axis Is an Important Regulator of Burkitt Lymphoma Cell Growth

The expression of several microRNAs (miRNAs) is known to be changed in Burkitt lymphoma (BL), compared to its normal counterparts. Although for some miRNAs, a role in BL was demonstrated, for most of them, their function is unclear. In this study, we aimed to identify miRNAs that control BL cell growth. Two BL cell lines were infected with lentiviral pools containing either 58 miRNA inhibitors or 44 miRNA overexpression constructs. Eighteen constructs showed significant changes in abundance over time, indicating that they affected BL growth. The screening results were validated by individual green fluorescent protein (GFP) growth competition assays for fifteen of the eighteen constructs. For functional follow-up studies, we focused on miR-26b-5p, whose overexpression inhibited BL cell growth. Argonaute 2 RNA immunoprecipitation (Ago2-IP) in two BL cell lines revealed 47 potential target genes of miR-26b-5p. Overlapping the list of putative targets with genes showing a growth repression phenotype in a genome-wide CRISPR/Cas9 knockout screen, revealed eight genes. The top-5 candidates included EZH2, COPS2, KPNA2, MRPL15, and NOL12. EZH2 is a known target of miR-26b-5p, with oncogenic properties in BL. The relevance of the latter four targets was confirmed using sgRNAs targeting these genes in individual GFP growth competition assays. Luciferase reporter assay confirmed binding of miR-26b-5p to the predicted target site for KPNA2, but not to the other genes. In summary, we identified 18 miRNAs that affected BL cell growth in a loss- or gain-of-function screening. A tumor suppressor role was confirmed for miR-26b-5p, and this effect could at least in part be attributed to KPNA2, a known regulator of OCT4, c-jun, and MYC

MiR-378a-3p Is Critical for Burkitt Lymphoma Cell Growth

Simple Summary MicroRNAs (miRNAs) are small RNAs that regulate expression of specific target genes. We observed elevated levels of miR-378a-3p in Burkitt lymphoma (BL) and studied its role in the pathogenesis of BL. Inhibition of miR-378a-3p reduced growth of BL cells, confirming its significance in BL. Identification of BL specific target genes of miR-378a-3p revealed four candidates. For two of them, MNT and IRAK4, miR-378a-dependent regulation was confirmed at the protein level. Overexpression of MNT and IRAK4 in BL cell lines resulted in a similar effect as observed upon miR-378a-3p inhibition, suggesting their involvement in the growth regulatory role of miR-378a-3p. MicroRNAs (miRNAs) are small RNA molecules with important gene regulatory roles in normal and pathophysiological cellular processes. Burkitt lymphoma (BL) is an MYC-driven lymphoma of germinal center B (GC-B) cell origin. To gain further knowledge on the role of miRNAs in the pathogenesis of BL, we performed small RNA sequencing in BL cell lines and normal GC-B cells. This revealed 26 miRNAs with significantly different expression levels. For five miRNAs, the differential expression pattern was confirmed in primary BL tissues compared to GC-B cells. MiR-378a-3p was upregulated in BL, and its inhibition reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for MAX Network Transcriptional Repressor (MNT), Forkhead Box P1 (FOXP1), Interleukin 1 Receptor Associated Kinase 4 (IRAK4), and lncRNA Just Proximal To XIST (JPX), and by Western blot for IRAK4 and MNT. Overexpression of IRAK4 and MNT phenocopied the effect of miR-378a-3p inhibition. In summary, we identified miR-378a-3p as a miRNA with an oncogenic role in BL and identified IRAK4 and MNT as miR-378a-3p target genes that are involved in its growth regulatory role

Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma

Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth /apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth-and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1. (C) 2018 The Author(s) Published by S. Karger AG, Base

Q & A on alternative social networking services

Q & A with Geert Lovin

Proliferation-promoting roles of linear and circular PVT1 are independent of their ability to bind miRNAs in B-cell lymphoma

Plasmacytoma Variant Translocation 1 (PVT1) is a long non-coding RNA located at 8q24.21 immediately downstream of MYC. Both the linear and circular PVT1 transcripts contribute to cancer pathogenesis by binding microRNAs. However, little is known about their roles in B-cell lymphoma. Here we studied their expression patterns, role in growth, and ability to bind miRNAs in B-cell lymphoma. Linear PVT1 transcripts were downregulated in B-cell cell lymphoma lines compared to germinal center B cells, while circPVT1 levels were increased. Two Hodgkin lymphoma cell lines had a homozygous deletion including the 5' region of the PVT1 locus, resulting in a complete lack of circPVT1 and 5' linear PVT1 transcripts. Inhibition of both linear and circular PVT1 decreased growth of Burkitt lymphoma, while the effects on Hodgkin lymphoma and diffuse large B cell lymphoma were less pronounced. Overexpression of circPVT1 promoted growth of B-cell lymphoma lacking or having low endogenous circPVT1 levels. Contrary to other types of cancer, linear and circular PVT1 transcripts did not interact with miRNAs in B-cell lymphoma. Overall, we showed an opposite expression pattern of linear and circular PVT1 in B-cell lymphoma. Their effect on growth was independent of their ability to bind miRNAs.</p

Cell plasticity in wound healing:paracrine factors of M1/M2 polarized macrophages influence the phenotypical state of dermal fibroblasts

<p>Background: Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs).</p><p>Results: HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production.</p><p>Conclusions: Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair.</p>

Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFN gamma) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages. (C) 2011 Elsevier GmbH. All rights reserved

Tubular engraftment and myofibroblast differentiation of recipient-derived cells after experimental kidney transplantation

Background. In human renal allografts, recipient-derived cells engrafted in various kidney substructures, have been detected in the long term after transplantation. Here we investigated tubular engraftment and myofibroblast differentiation of recipient-derived cells at short term after experimental kidney transplantation, during a previously described window of regeneration and possible onset of renal interstitial fibrosis. Methods. Fisher (F344, syngeneic) and Dark Agouti (DA, allogeneic) kidneys were transplanted into F344-hPAP transgenic recipient rats, which allowed tracing of recipient-derived cells in nontransgenic donor kidneys. We evaluated tubular engraftment and myofibroblast differentiation of recipient-derived cells on day 14 after kidney transplantation. Results. Kidney transplantation resulted in tubular engraftment of recipient- derived cells. After allogeneic kidney transplantation, 9.7% of tubular cross-sections contained at least one recipient- derived cell, which represented a significant increase in comparison to syngeneic transplantation (4.0%, P <0.05). Moreover, recipient-derived myofibroblasts were present in the renal interstitium of the transplanted kidney. These cells contributed 39% of the total interstitial myofibroblast population in allografts, which was comparable to the syngeneic situation (28%, P=0.25). Conclusions. In a defined early window of regeneration and possible onset of renal interstitial fibrosis after kidney transplantation, rejection-associated injury, superimposed on ischemic damage, increases tubular engraftment of recipient-derived cells, although it does not affect their relative contribution to the renal interstitial myofibroblast population