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, 9 (102), 37616-37626

piRNA-8041 Is Downregulated in Human Glioblastoma and Suppresses Tumor Growth in vitro and in vivo

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piRNA-8041 Is Downregulated in Human Glioblastoma and Suppresses Tumor Growth in vitro and in vivo

Daniel I Jacobs et al. Oncotarget.

Abstract

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that partner with PIWI proteins to protect germline tissues from destabilizing transposon activity. While the aberrant expression of PIWI proteins has been linked with poor outcomes for many cancers, less is known about the expression or function of piRNAs in cancer. We performed array-based piRNA expression profiling in seven pairs of normal brain and glioblastoma multiforme (GBM) tissue specimens, and identified expression of ~350 piRNAs in both tissues and a subset with dysregulated expression in GBM. Over-expression of the most down-regulated piRNA in GBM tissue, piR-8041, was found to reduce glioma cell line proliferation, induce cell cycle arrest and apoptosis, and inhibit cell survival pathways. Furthermore, pre-treatment with piR-8041 significantly reduced the volume of intracranial mouse xenograft tumors. Taken together, our study reveals reduced expression in GBM of piR-8041 and other piRNAs with tumor suppressive properties, and suggests that restoration of such piRNAs may be a potential strategy for GBM therapy.

Keywords: GBM; PIWI-interacting RNA; glioma; piRNA.

Conflict of interest statement

CONFLICTS OF INTEREST The authors have declared that no conflicts of interest exists.

Figures

Figure 1
Figure 1. piRNA expression profiling results and confirmation of piR-8041 underexpression in GBM relative to normal brain tissue
(A) Results of array-based piRNA expression profiling in GBM relative to normal pooled tissue specimens. piRNAs with detectable expression levels are plotted according to average log2(signal intensity) in each tissue type. piR-8041 and three additional piRNAs examined in subsequent cell proliferation analyses are labeled (piR-20249, piR-54022, piR-15988), as well as two piRNAs previously shown to be dysregulated in cancer (piR-651, piR-823). (B) Validation of piR-8041 expression levels in individual normal vs. tumor tissue specimens by qPCR. Data are presented as log2(piR-8041 expression level) relative to small RNA U6 expression; lines denote mean expression level by tissue type. (C) Measurement of piR-8041 expression in normal human astrocytes (NHA) and glioma cell lines U87 and A172 by qPCR. **, P < 0.01; ***, P < 0.001; error bars denote standard deviation of triplicate measurements.
Figure 2
Figure 2. Reduction of GBM cell proliferation by piR-8041 and other GBM-underexpressed piRNAs
Growth inhibition was specific to GBM-underexpressed piRNAs and glioma cell lines, piR-8041 reduced long-term colony formation, and growth inhibition was enhanced with a secondary treatment. (A) U87 cell proliferation following transfection of piRNAs underexpressed in tumor relative to normal brain tissue (fold-changes noted in figure legend) or piRNAs equivalently expressed in tumor and normal brain tissue (no association). Values denote ratio of color development after MTS exposure of piRNA-treated cells relative to negative control (NC)-treated cells; dotted line represents equivalent cell viability after piRNA or negative control RNA exposure. (B) NHA, A172, and U87 cell proliferation following piR-8041 upregulation. Values denote relative viability of piR-8041 vs. NC-treated cells and statistical significance was assessed by the deviation from NC treatment, denoted by the dotted line. (C) U87 colonies formed in soft agar 21 days after piR-8041 or NC transfection. Colonies were counted using ImageJ software; representative images are presented. (D) U87 cell viability at six days following one (day 0 only) or two (day 0 and day 3) piR-8041 treatments. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; error bars denote standard deviation of triplicate experiments for all figures.
Figure 3
Figure 3. piR-8041 upregulation impacts expression of genes related to cellular survival, stress, and other glioma-relevant functions
(A) List of biological functions statistically significantly enriched among genes differentially expressed by piR-8041 upregulation in U87 cells. Bars indicate the number of genes impacted with a particular functional annotation; diamonds denote the log-transformed FDR-adjusted P-values (dotted line indicates an FDR-adjusted P-value of 0.05). (B) Illustration of top network of differentially expressed transcripts, related to “decreased cell viability of connective tissue cells” and “decreased synthesis of protein” following piR-8041 treatment of U87 cells. Red and green shading denote transcript over- and under-expression relative to negative control after piR-8041 upregulation, respectively, with color intensity corresponding to degree of change, and blue shading denotes predicted signaling pathway inhibition. Solid lines and dotted lines indicate direct and indirect relationships, respectively.
Figure 4
Figure 4. piR-8041 treatment of U87 cells inhibits cell cycle progression by approximately 25% and induces apoptosis
(A) Cell cycle distribution 48 hours post-piR-8041 or NC-treatment. Cell cycle phases were determined by flow cytometric analysis of DNA content by staining with propidium iodide. Representative cell cycle distributions of NC- and piR-8041-treated U87 cells are shown at right with modeled phase fractions superimposed on DNA content histograms. (B) Proportions of U87 cells in early or late apoptosis/necrosis 48 hours post-piR-8041 or NC-treatment. Early apoptotic cells were defined as those stained with Annexin V but excluding PI, late apoptotic/necrotic were cells stained with both probes; representative plots are shown at right. NS, not significant; *, P < 0.05; **, P < 0.01; error bars denote standard deviation of triplicate experiments.
Figure 5
Figure 5. piR-8041 reduces U87 cell growth by nearly 50% 10 days after treatment in an orthotopic xenograft model
(A) Bioluminescence measurements of luciferase-expressing intracranial tumors at multiple timepoints. Luminescence intensity was measured as a proxy for tumor volume using an IVIS SpectrumCT Imaging System following intravitreal luciferin injection. Statistical significance was assessed by Student's t-test between treatment conditions at each time point; associated P-values are presented along with average piR-8041-treated tumor intensity as a percentage of control intensity. (B) Images of representative mice from each treatment group on day 10 after tumor implantation. Colors correspond to the luminescence scale presented at right, with red and blue coloring representing high and low luminescence intensity, respectively.

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