Generation of BCBL-1 and BC-3 cells with stable HIF-1α knockdown.

<p><b>(A)</b> Protein levels of HIF-1α in nuclear extracts of BJAB, BCBL-1, and BC-3 cells measured by Western blot analysis after 24 hours in normoxia. Tata-binding protein (TBP) is used as a loading control. BCBL-1 and BC-3 cells were transduced with lentivirus encoding shRNA to HIF-1α or Scrambled (Scr) RNA and stable cell lines were generated with puromycin selection. Total RNA and nuclear protein extracts were extracted from the cells to confirm the status of the knockdown. (<b>B</b>) mRNA levels of HIF-1α measured by RT-qPCR after 48 hours in normoxia(N) or hypoxia(H). mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under normoxia. (<b>C and D</b>) Protein levels of HIF-1α measured by Western blot analysis of nuclear extracts after 24 hours in culture. β-actin is shown as a loading control. (<b>C</b>) Normoxic levels of HIF-1α levels in the absence or presence of 50μM cobalt chloride (CoCl₂), a hypoxia mimic that prevents oxygen-induced degradation of HIF-1α. (<b>D</b>) HIF-1α levels under normoxia or hypoxia.</p

Effects of HIF-1α inhibitor PX-478 on PELs.

<p>(<b>A</b>) HIF-1α mRNA levels in BCBL-1 cells 24 hours after treatment with various concentrations of PX-478, normalized to 18S internal control and expressed as fold changes compared to no PX-478 control cells. (<b>B)</b> Proliferation rate of PEL cell lines BCBL-1, BC-3, JSC-1, BC-1, and BC-2 and Burkitt’s lymphoma (BL) cell lines BJAB and CA46 measured using the MTS assay 72 hours after treatment with indicated concentrations of PX-478, expressed as fold changes compared to no PX-478 control cells. (<b>C)</b> Growth rates of the PEL and BL cells treated with 0 or 10 μM PX-478. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between untreated and inhibitor-treated cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p

Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

<div><p>Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.</p></div

Effect of HIF-1α knockdown on the expression of KSHV miRNAs.

<p><b>(A)</b> Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. <b>(B)</b> Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05 (2-tailed t-test).</p

miRNA-mRNA pairs found inversely correlated and validated in the literature.

<p>This table shows the association between miRNAs and mRNAs that were significantly dysregulated in SLKK cells compared to SLK cells. Only the miRNA-mRNA pairs experimentally validated in the literature are shown here. The majority of the targets were identified through IPA-integrated TarBase v5.0 and others independently.</p><p>miRNA-mRNA pairs found inversely correlated and validated in the literature.</p

Differential expression of miRNAs and their known or predicted mRNA targets.

<p>A. miRNA expression in latently infected SLKK cells compared to uninfected SLK cells and the mRNA expression of their corresponding targets (Taqman and qRT-PCR assays). miRNAs and mRNAs are shown in light and dark grey, respectively. miR-409-3p targets fibrinogen beta (FGB) and radixin (RDX). miR-409-5p is predicted to target MDM2. miR-708-5p targets caspase-2 (CASP2) and is predicted to target leukemia inhibitor factor (LIF). P-values were calculated using Student t-test. *, ** and *** indicate P <0.05, 0.01 and 0.001, respectively. B. Effect of miRNA transfection on target regulation. Scramble control (miR-scramble) and miRNA mimics for miR-409-3p, miR-409-5p and miR-708-5p were transfected in either SLK or SLKK cells. Quantitative real-time polymerase chain reaction revealed the target expression for these miRNAs and the relative value is presented as the mean ± standard deviation based on 3 independent experiments. Statistical analysis and its representation are as in Fig 4A.</p

Expression of miRNAs and mRNAs in the analyzed libraries.

<p>A. Volcano plot of differentially expressed human mature miRNAs in KSHV-infected versus uninfected cells. Vertical red lines indicate the threshold for a relative expression fold change (FC) of 2 or -2 fold compared to uninfected controls. The horizontal red line represents the threshold of a 0.05 P-value. Thus, the blue points lying in the top right and top left sectors are significantly up-regulated and down-regulated, respectively, in KSHV-positive versus KSHV-negative cells (<i>P</i> <0.05, FC ≥2 or ≤-2). Selected miRNAs that have been validated by qRT-PCR are labeled. B. Volcano plot of differentially expressed mRNAs in KSHV-infected versus uninfected cells. The plot is depicted as in Fig 2A, with vertical and horizontal red lines similarly representing the thresholds of a fold-change of 2 or -2 fold, and of a false-discovery rate (FDR) of 0.05 respectively. C. Top 15 repressed and induced miRNAs in KSHV-positive vs. uninfected SLK cells with <i>P</i> <0.05 and medium to high miRNA expression (threshold of a mean miR count of 10 or more across all replicates).</p

Effect of HIF-1α knockdown on the expression of KSHV latent genes in PELs.

<p>(<b>A</b>) Schematic of multicistronic KSHV latency locus showing the location of latent genes and hypoxia-response elements (HREs) in the promoter region. (<b>B</b>) mRNA levels of latent genes in BCBL-1 cells within and (<b>C</b>) outside of the latency locus as measured by RT-qPCR and normalized to that of 18S RNA. RNA levels for each gene are expressed as fold change relative to shScr cells under normoxia (N). Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01 (2-tailed t-test).</p

HIF-1α knockdown leads to reduced lytic replication of KSHV.

<p><b>(A)</b> mRNA levels of RTA, vIL-6, and ORF26 (a late lytic gene) of KSHV in BCBL-1 cells measured by RT-qPCR. Error bars represent standard deviation from at least 3 experiments. (<b>B)</b> Western blot showing protein levels of intracellular RTA, vIL6, and β-actin in the cell lysates as well as secreted vIL6 in the supernatant after 48 hours in normoxia (N) or hypoxia (H). (<b>C)</b> Protein levels of ORF45 in concentrated virus particles released in the supernatants after 72 hours. *Unspecific band. Western blots were done on lysates from three independent experiments and representative blots are shown.</p

Effect of HIF-1α suppression on growth of PELs.

<p><b>(A)</b> Growth rate of shScr or shHIF-1 BCBL-1 and BC-3 cells measured by counting live cells every 24 hours in normoxia. (<b>B)</b> Proliferation rate of shScr or shHIF-1 cells measured by MTS assay at 72 hours in normoxia using Promega’s CellTiter 96 Aqueous One Solution assay, which measures the amount of NADH or NADPH produced by metabolically active cells. (<b>C)</b> Colony forming efficiency of shHIF-1 cells relative to shScr cells. Error bars represent standard deviations from at least 3 independent experiments. Results shown in B and C are fold changes compared to shScr cells. Statistically significant differences between shScr and shHIF-1 cells are indicated. *<i>P</i> ≤0.05, **<i>P</i> ≤ 0.01.</p