CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.

BackgroundLong non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.ResultsWe use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.ConclusionsThese studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy

Arabinosylated Lipoarabinomannan - mediated protection in Visceral Leishmaniasis through up-regulation of toll-like receptor 2 signaling: an immunoprophylactic approach

Visceral leishmaniasis is characterized by severe immunosuppression of the host cell, resulting in loss of the proinflammatory response. Toll-like receptor 2 (TLR2) is involved in myriad disease forms, including visceral leishmaniasis. During Leishmania donovani infection, the parasite modulates TLR2 to suppress interleukin 12 production, indicating the possible involvement of TLR2 in regulation of the immune response against L. donovani infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immunomodulatory properties and induces proinflammatory responses via induction of TLR2-mediated signaling. Here, we found that pretreatment of L. donovani-infected macrophages with Ara-LAM caused a significant increase in TLR2 expression along with the activation of TLR2-mediated downstream signaling, facilitating active nuclear translocation of nuclear factor κB. These events culminated in up-regulation of the proinflammatory response, which was abrogated by treatment with TLR2-specific small interfering RNA. In vivo experiments were also suggestive of Ara-LAM playing a long-term protective role. This study demonstrates that Ara-LAM confers protection against leishmanial pathogenesis via TLR2 signaling-mediated induction of the proinflammatory response

miRNA-independent function of long noncoding pri-miRNA loci.

Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29Δ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms

<i>Mw</i> induces the nitrite generation in <i>Leishmania</i><i> </i><i>donovani</i> infected murine macrophages (A–C)

<p>(A) Uninfected (UIM) and infected (IM) macrophages were treated with <i>Mw</i> (10⁷cells/ml), suboptimal dose of AmpB (0.1 µg/ml) and <i>Mw</i> plus AmpB (0.1 µg/ml). After 48 hours the cell free supernatant were collected and nitrate generation were measured in different sets. (B) mRNA expression of iNOS2 from similar experiments were determined by Real Time -PCR, (C) Macrophages cultured in chambered cover slides were treated with or without L-NMMA (0.4 mmol/L) for 1 h prior to infection followed by treatment with Mw and AmpB for 20 h and 44 h. After that, cover slips were stained with Giemsa and assessed for intracellular parasites number. L-NMMA significantly inhibited parasite killing activity of <i>Mw</i> and the similar type of inhibition was also observed in case of combination of <i>Mw</i> plus AmpB treatment. Results are presented as mean values. The experiment was repeated 3 times, yielding similar results and data were expressed as mean ± SD. ^*P<0.001, ^**P<0.01 compared with infected macrophages and ^***P<0.05 compared with <i>Mw</i> treated macrophages; paired two-tailed Student’s t-test.</p

Primers used in Real Time -PCR assay.

<p>Primers used in Real Time -PCR assay.</p

<i>Mw</i> significantly reduces the intracellular parasitic load (A–D).

<p>Peritoneal macrophages, isolated from BALB/c mice, were cultured in 8-chambered glass cover slides with complete RPMI 1640 media followed by infection with <i>L. donovani</i> promastigotes (macrophage: parasitic ratio of 1∶10) for 4 h. Macrophages were treated with different doses of <i>Mw</i> (A) or AmpB (B) for 20 h and 44 h. Intracellular parasites were counted per 100 macrophages after Giemsa staining. Similarly, in a separate experiment, macrophages were infected with <i>L. donovani</i> promastigotes followed by treatment with <i>Mw</i> (10⁷ cells/ml) along with different doses of AmpB for 20 h and 44 h as indicated in the (C) & (D). Intracellular parasites were counted per 100 macrophages after Giemsa staining. <b>Determination of noncytotoxic dose of </b><b><i>Mw</i></b><b> cell wall and it effect on the parasitic burden in </b><b><i>L. donovani</i></b><b>-infected murine peritoneal macrophages </b><b>(E–F).</b> Uninfected (UIM) and infected (IM) macrophages were subjected to <i>Mw</i> cell wall treatment at specified doses (0.1–20 µg/ml) After 48 h of incubation, cell viability assay was performed using the MTT method (E). Infected (IM) macrophages were treated with different doses of <i>Mw</i> cell wall (2–7 µg/ml) for 20 h and 44 h (F). Intracellular parasites were counted per 100 macrophages after Giemsa staining. The experiment was repeated 3 times, yielding similar results and data were expressed as mean ± SD. ^*P<0.001, ^**P<0.01 compared with infected macrophages and ^***P<0.05 compared with <i>Mw</i> treated macrophages; paired two-tailed Student’s t-test.</p

Treatment with <i>Mw</i> or <i>Mw</i> plus AmpB (sub) causes complete clearance of hepatic and splenic parasitic burden of infected BALB/c mice (A–B).

<p>BALB/c and IL-12 knockout mice were infected with <i>L. donovani</i>, followed by treatment with either phosphate buffered saline (PBS; control), <i>Mw</i> (10⁸cells/100 µl), suboptimal dose of AmpB (2.1 mg/kg body weight) and <i>Mw</i> plus AmpB (suboptimal). Mice were sacrificed on day, 56 after infection. Untreated, infected mice were used as controls. Levels of parasitic burden in liver (A) and spleen (B) samples were expressed in Leishman Donovan Units (LDU). Data were expressed as mean ± SD (n = 4 mice per group) ^*P<0.001, ^**P<0.01 compared with infected mice and ^***P<0.05 compared with <i>Mw</i> treated infected mice; paired two-tailed Student’s t-test.</p

<i>Mw</i> induced Th1 response and NO generation in <i>L. donovani</i> infected BALB/c mice (A–F).

<p>Splenocytes from infected and differently–treated mice, as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040265#pone-0040265-g006" target="_blank">figure 6</a> legend, were isolated on 56 days after infection, plated aseptically (2×10⁵ cells/well), and stimulated with 10 µg/ml soluble leishmanial antigen (SLA) for 48 h. Interferon γ (IFN- γ) (A), nitric oxide (B), Tumor necrosis factor α (TNF-α) (C), Interleukin 10 (IL-10) (D) Interleukin 12 (IL-12) (E) and Tumor growth factor β (TGF- β) (F) from the culture supernatants of the indicated treatment groups were determined by enzyme-linked immunosorbent assay and the Nitric Oxide Colorimetric Assay kit, respectively. Real-Time polymerase chain reaction (PCR) analysis was performed from similar experimental sets to determine proinflammatory (IFN-γ, IL-12p70, TNF-α), anti-inflammatory cytokines (IL-10, TGF-β) and iNOS2 mRNA expression (G, H). Results are from three independent experiments. Data are means ± standard deviations of values from 3 independent experiments that yielded similar results^*P<0.001, ^**P<0.01 compared with infected mice and ^***P<0.05 compared with <i>Mw</i> treated infected mice; paired two-tailed Student’s t-test.</p

IL-12 is critical for <i>Mw</i> and suboptimal dose of AmpB mediated protection against <i>Leishmania donovani</i> infection (A–F).

<p>(A) Peritoneal macrophages, isolated from BALB/c and IL-12p40^−/− mice, were cultured in eight chambered cover slides and infected with <i>L. donovani</i>. Infected macrophages were treated with <i>Mw</i> (10⁷/ml) or <i>Mw</i> plus AmpB (0.1 µg/ml) for 20 h and 44 h and intracellular parasites were counted per 100 macrophages after Giemsa staining. The experiment was repeated four times, yielding similar results and data were expressed as mean ± SD. (B) Uninfected or Leishmania infected BALB/c and IL-12p40^−/− peritoneal macrophages (10⁶cells/ml) were treated with <i>Mw</i> (10⁷cells/ml), AmpB (0.1 µg/ml), or <i>Mw</i> plus AmpB for 48 h. Cell-free supernatants were assayed for nitrite generation by Griess method as described in the Material method section. (C) Uninfected or <i>Leishmania</i> infected BALB/c and IL-12p40^−/− peritoneal macrophages (2×10⁶cells/ml) treated with <i>Mw</i> (10⁷cells/ml), AmpB (0.1 µg/ml), or <i>Mw</i> plus AmpB, for 20 h. Cell-free supernatants were collected for estimation of TNF-α, TGF-β, and IL-10 by sandwich ELISA. (F) Total RNA was extracted from similarly treated uninfected or <i>Leishmania</i> infected BALB/c and IL-12p40^−/− peritoneal macrophages using TRIZOL and Real Time -PCR was performed to study mRNA expression of different pro-inflammatory and anti-inflammatory cytokines. The experiment was repeated 3 times, yielding similar results and data were expressed as mean ± SD. ^*P<0.001, ^**P<0.01 compared with infected macrophages and ^***P<0.05 compared with <i>Mw</i> treated macrophages; paired two-tailed Student’s t-test.</p