microRNA dependent and independent deregulation of long non-coding RNAs by an oncogenic herpesvirus

<div><p>Kaposi’s sarcoma (KS) is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL). In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs). The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA) expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.</p></div

LEVERAGING BIOLOGICAL REPLICATES TO IMPROVE ANALYSIS IN CHIP-SEQ EXPERIMENTS

ChIP-seq experiments identify genome-wide profiles of DNA-binding molecules including transcription factors, enzymes and epigenetic marks. Biological replicates are critical for reliable site discovery and are required for the deposition of data in the ENCODE and modENCODE projects. While early reports suggested two replicates were sufficient, the widespread application of the technique has led to emerging consensus that the technique is noisy and that increasing replication may be worthwhile. Additional biological replicates also allow for quantitative assessment of differences between conditions. To date it has remained controversial about how to confirm peak identification and to determine signal strength across biological replicates, particularly when the number of replicates is greater than two. Using objective metrics, we evaluate the consistency of biological replicates in ChIP-seq experiments with more than two replicates. We compare several approaches for binding site determination, including two popular but disparate peak callers, CisGenome and MACS2. Here we propose read coverage as a quantitative measurement of signal strength for estimating sample concordance. Determining binding based on genomic features, such as promoters, is also examined. We find that increasing the number of biological replicates increases the reliability of peak identification. Critically, binding sites with strong biological evidence may be missed if researchers rely on only two biological replicates. When more than two replicates are performed, a simple majority rule (>50% of samples identify a peak) identifies peaks more reliably in all biological replicates than the absolute concordance of peak identification between any two replicates, further demonstrating the utility of increasing replicate numbers in ChIP-seq experiments

KSHV miRNAs directly bind to and downregulate host lncRNAs.

<p><b>(A)</b> Uninfected TIVE cells were transfected with 5 nM final concentration of miRNA mimic pools (<i>Loc541472</i>: miR-K12-1, K12-6-5p; <i>CD27-AS1</i>: miR-K12-1*, K12-11*; <i>RP11-438-N16</i>.<i>1</i>: miR-K12-1*, K12-8*, K12-11*; <i>Linc00607</i>: miR-K12-2*, K12-11*). Relative expression levels of target lncRNAs were analyzed 48 h post-transfection using qRT-PCR. The bar graphs show the mean values ± SEM after normalization to GAPDH (n = 3). <b>(B)</b> Biotinylated miRNA mimics of miR-K12-6-5p and miR-K12-11* were transfected into uninfected TIVE-ExLTC cells (5 nM final concentration) and were pulled down 24 h later. Target lncRNAs were analyzed using qRT-PCR. siGLO pulldown was used a negative control. The bar graphs show the mean values ± SEM after normalization to input (n = 3). p-values: * < 0.01; ** < 0.005; *** < 0.0005; and **** <0.0001.</p

LncRNA ANRIL is targeted by both KSHV miRNAs and latency proteins.

<p>All bar graphs show the mean values ± SEM after normalization to GAPDH (n = 3), unless specified otherwise. <b>(A)</b> ANRIL expression in Uninfected, wt-KSHV-infected and Δcluster-KSHV-infected cells measured by qRT-PCR. <b>(B)</b> Uninfected and wt-KSHV-infected TIVE cells were transfected with pcDNA3.1-ANRIL and relative over-expression of ANRIL was measured using qRT-PCR. LSD-1 was used a control to verify comparable transfection efficiencies of uninfected and infected cells. Y-axis is calculated as the ratio of fold-overexpression observed in wt-KSHV infected cells to the fold-overexpression observed in uninfected cells. Overexpressions were normalized to any expression changes observed by transfecting empty vector, which is thus set at one. <b>(C)</b> Uninfected TIVE cells were transfected with 5 nM final concentration of miRNA mimic pool (miR-K12-1*, K12-6-5p, K12-2* and K12-11*). Relative expression level of ANRIL was analyzed 48 h post-transfection using qRT-PCR. (<b>D</b>) Biotinylated miRNA mimics of miR-K12-6-5p and miR-K12-11* were transfected into uninfected TIVE cells (5 nM final concentration) and were pulled down 24 h later. ANRIL expression was analyzed using qRT-PCR. siGLO pulldown was used as a negative control. The data were normalized to input. <b>(E)</b> ANRIL expression in Uninfected, wt-KSHV-infected and Δall-KSHV-infected cells measured by qRT-PCR (n = 2). <b>(F)</b> HeLa cells were transfected with latency gene(s) (LANA, vCyclin, vFLIP, Kaposin or vCyclin + Kaposin) expressed from pcDNA3.2 vector. ANRIL expression was analyzed 72 h post-transfection using qRT-PCR. p-values: * < 0.05; ** < 0.01; *** < 0.005; **** < 0.0005; ***** < 10⁻⁴ and n.s. = not significant.</p

Tumor suppressor lncRNA MEG3 is targeted by KSHV miRNAs.

<p>All bar graphs show the mean values ± SEM after normalization to GAPDH (n = 3), unless specified otherwise. <b>(A)</b> MEG3 expression in Uninfected, wt-KSHV-infected and Δcluster-KSHV-infected cells measured by qRT-PCR. <b>(B)</b> Uninfected TIVE cells were transfected with 5 nM final concentration of miRNA mimic pool (miR-K12-5, K12-6-5p and K12-8*). Relative expression level of MEG3 was analyzed 48 h post-transfection using qRT-PCR. <b>(C)</b> Biotinylated miRNA mimic of miR-K12-6-5p was transfected into uninfected TIVE cells (5 nM final concentration) and was pulled down 24 h later. MEG3 expression was analyzed using qRT-PCR. siGLO pulldown was used as a negative control. The data were normalized to input. p-values: * < 0.05; ** < 0.01; *** < 0.005; **** < 0.0005; and ***** < 10⁻⁴.</p

Expression profiling of wt-KSHV and Δcluster-KSHV infected endothelial cells.

<p><b>(A)</b> Latency associated region of wt-KSHV in a Bac16 backbone. The region deleted in the Δcluster-KSHV virus is highlighted. <b>(B)</b> Heatmap of unsupervised hierarchical clustering of the microarray samples in the ‘rescued’ category of genes (n = 3 technical replicates).</p

Summary of deregulated lncRNAs from the microarray analysis.

<p>Summary of deregulated lncRNAs from the microarray analysis.</p