Search

Supplementary Figure 3 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 12 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 4 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Methods, Figure Legends 1-13 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 9 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 3 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 8 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 2 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 5 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 7 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Figure 6 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 1 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

Supplementary Table 2 from MicroRNAs miR-199a-5p and -3p Target the Brm Subunit of SWI/SNF to Generate a Double-Negative Feedback Loop in a Variety of Human Cancers

The chromatin remodeling complex SWI/SNF is an important epigenetic regulator that includes one Brm or BRG1 molecule as catalytic subunit. Brm and BRG1 do not function identically, so this complex can regulate gene expression either positively or negatively, depending on the promoter to which it is recruited. Notably, Brm attenuation due to posttranscription suppression occurs often in human tumor cells, in which this event contributes to their oncogenic potential. Here, we report that the 3′-untranslated region of Brm mRNA has two sites that are efficiently targeted by the microRNAs miR-199a-5p and -3p, revealing a novel mechanism for modulation of Brm-type SWI/SNF activity. Computational mapping of the putative promoter region of miR-199a-2 (miPPR-199a-2) has defined it as the major contributing genetic locus for miR-199a-5p and-3p production in these tumor cell lines. We validated this predicted region by direct promoter analysis to confirm that Egr1 is a strong positive regulator of the miR-199a-2 gene. Importantly, we also showed that Egr1, miR-199a-5p, and miR-199a-3p are expressed at high levels in Brm-deficient tumor cell lines but only marginally in Brm-expressing tumor cells. Finally, we also obtained evidence that Brm negatively regulates Egr1. Together, our results reveal that miR-199a and Brm form a double-negative feedback loop through Egr1, leading to the generation of these two distinct cell types during carcinogenesis. This mechanism may offer a partial explanation for why miR-199a-5p and -3p have been reported to be either upregulated or downregulated in a variety of tumors. Cancer Res; 71(5); 1680–9. ©2010 AACR.

Non-coding RNAs (ncRNAs) play important roles in host-pathogen interactions; oncogenic viruses like Kaposi’s sarcoma herpesvirus (KSHV) employ ncRNAs to establish a latent reservoir and persist for the life of the host. We previously reported that KSHV infection alters a novel class of RNA, circular RNAs (circRNAs). CircRNAs are alternative splicing isoforms and regulate gene expression, but their importance in infection is largely unknown. Here, we showed that a human circRNA, hsa_circ_0001400, is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. The induction of circRNAs including circ_0001400 by KSHV is co-transcriptionally regulated, likely at splicing. Consistently, screening for circ_0001400-interacting proteins identified a splicing factor, PNISR. Functional studies using infected primary endothelial cells revealed that circ_0001400 inhibits KSHV lytic transcription and virus production. Simultaneously, the circRNA promoted cell cycle, inhibited apoptosis, and induced immune genes. RNA-pull down assays identified transcripts interacting with circ_0001400, including TTI1 , which is a component of the pro-growth mTOR complexes. We thus identified a circRNA that is pro-growth and anti-lytic replication. These results support a model in which KSHV induces circ_0001400 expression to maintain latency. Since circ_0001400 is induced by multiple viruses, this novel viral strategy may be widely employed by other viruses.

Non-coding RNAs (ncRNAs) play important roles in host-pathogen interactions; oncogenic viruses like Kaposi sarcoma herpesvirus (KSHV) employ ncRNAs to establish a latent reservoir and persist for the life of the host. We previously reported that KSHV infection alters a novel class of RNA, circular RNAs (circRNAs). CircRNAs are alternative splicing isoforms and regulate gene expression but, their importance in infection is largely unknown. Here, we showed that a human circRNA, hsa_circ_0001400, is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. The induction of circRNAs including circ_0001400 by KSHV is co-transcriptionally regulated, likely at splicing. Consistently, screening for circ_0001400-interacting proteins identified a splicing factor, PNISR. Functional studies using infected primary endothelial cells revealed that circ_0001400 inhibits KSHV lytic transcription and virus production. Simultaneously, the circRNA promoted cell cycle, inhibited apoptosis, and induced immune genes. RNA-pulldown assays identified transcripts interacting with circ_0001400, including TTI1, which is a component of the pro-growth mTOR complexes. We thus identified a circRNA that is pro-growth and anti-lytic replication. These results support a model in which KSHV induces circ_0001400 expression to maintain latency. Since circ_0001400 is induced by multiple viruses, this novel viral strategy may be widely employed by other viruses.AUTHOR SUMMARYCircular RNAs (circRNAs) are single-stranded, closed-circular RNAs. They coincide with linear mRNAs as alternative splice isoforms. CircRNAs binds to other RNAs or proteins to regulate their abundance or functions. We previously showed that specific human circRNAs are differentials regulated upon infection with an oncogenic DNA virus, Kaposi’s sarcoma herpesvirus (KSHV). Functions of such circRNAs for virus infections were largely elusive. Here, we identified that one of circRNAs, hsa_circ_0001400 controls both viral and human gene expression so that infected human cells have less virus production but better cell growth. KSHV, like other herpesviruses, has two phases: latent and lytic cycle. The functions of circ_0001400 suggest it biases the cells to latent cycle during which, unlike lytic phase, viruses express only limited number of genes to survive and new viruses are not produced. Since oncogenic viruses like KSHV mainly stay at latent phase for the life of the host, our results suggest that the virus utilized circRNAs to switch to latent phase after the infection. Infection of other pathogenic viruses like Epstein-Barr virus and human cytomegalovirus were also found to induce circ_0001400. The circRNA may be thus controlling infection of various viruses.

Kaposi sarcoma (KS), caused by Kaposi sarcoma herpesvirus (KSHV), is a multicentric tumor characterized by abnormal vasculature and proliferation of KSHV-infected spindle cells. KS commonly involves the skin but in severe cases KS can also involve the gastrointestinal tract (GI). Here, we sought to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions. Skin and GI KS were compared to normal matched samples using bulk RNA sequencing.Twenty-two paired samples of KS and normal tissue were obtained (skin (10 pairs) and GI (12 pairs)) from 19 patients with KS of whom 17 had concurrent HIV infection. Seven paired samples were from patients who had received prior KS therapy. Three patients provided both skin and GI samples at the same timepoint. These analyses identified 370 differentially expressed genes unique to cutaneous KS and 58 DEGs unique to GI KS compared to normal skin or GI tissues. Twenty-six differentially expressed genes overlapped between skin and GI KS, which included FLT4, which encodes for a VEGF-C and VEGF-D receptor, and STC1. KSHV infection of primary lymphatic endothelial cells (LECs) resulted in increased angiogenesis, and repression of STC1 or FLT4 inhibited angiogenesis. The analyses of KSHV expression from KS lesions identified certain lytic genes, specifically ORF75, that were consistently expressed, and these expression patterns differed from laboratory infection of LECs with KSHV and KSHV gene expression in PEL cell lines. This study demonstrates that complex patterns of gene expression are found in KS tissue that differ from the canonical latent/lytic programs seen in KSHV cell lines and also demonstrates differences in viral gene and clinically relevant host gene expression in skin and GI KS that may offer insights into the pathogenesis of these forms of KS.One sentence summaryKaposi sarcoma that manifests in the skin and gastrointestinal tracts differ by human and viral gene expression.

Oncogenic gammaherpesviruses express viral products during latent and lytic infection that block the innate immune response. Previously, we found that Kaposi’s sarcoma herpesvirus (KSHV/human herpesvirus-8) viral microRNAs (miRNAs) downregulate cholesterol biogenesis, and we hypothesized that this prevents the production of 25-hydroxycholesterol (25HC), a cholesterol derivative. 25HC blocks KSHV de novo infection of primary endothelial cells at a postentry step and decreases viral gene expression of LANA (latency-associated nuclear antigen) and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using RNA sequencing (RNA-Seq). We found that 25HC treatment inhibited KSHV gene expression and induced interferon-stimulated genes (ISGs) and several inflammatory cytokines (interleukin 8 [IL-8], IL-1α). Some 25HC-induced genes were partially responsible for the broadly antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited infection of primary B cells by a related oncogenic virus, Epstein-Barr virus (EBV/human herpesvirus-4) by suppressing key viral genes such as LMP-1 and inducing apoptosis. RNA-Seq analysis revealed that IL-1 and IL-8 pathways were induced by 25HC in both primary endothelial cells and B cells. We also found that the gene encoding cholesterol 25-hydroxylase (CH25H), which converts cholesterol to 25HC, can be induced by type I interferon (IFN) in human B cell-enriched peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral miRNAs target the cholesterol pathway to prevent 25HC production and subsequent induction of antiviral ISGs. Together, these results answer some important questions about a widely acting antiviral (25HC), with implications for multiple viral and bacterial infections. IMPORTANCE A cholesterol derivative, 25-hydroxycholesterol (25HC), has been demonstrated to inhibit infections from widely different bacteria and viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, its mechanism of activity is still not fully understood. In this work, we look at gene expression changes in the host and virus after 25HC treatment to find clues about its antiviral activity. We likewise demonstrate that 25HC is also antiviral against EBV, a common cancer-causing virus. We compared our results with previous data from antiviral screening assays and found the same pathways resulting in antiviral activity. Together, these results bring us closer to understanding how a modified form of cholesterol works against several viruses.