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Analysis of EZH2: micro-RNA network in low and high grade astrocytic tumors

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Abstract

Enhancer of Zeste homologue2 (EZH2) is an epigenetic regulator that functions as oncogene in astrocytic tumors, however, EZH2 regulation remains little studied. In this study, we measured EZH2 levels in low (Gr-II,DA) and high grade (Gr-IV,GBM) astrocytic tumors and found significant increased EZH2 transcript level with grade(median DA-8.5, GBM-28.9).However, a different trend was reflected in protein levels, with GBMs showing high EZH2 LI(median-26.5) compared to DA (median 0.3). This difference in correlation of EZH2 protein and RNA levels suggested post-transcriptional regulation of EZH2, likely mediated by miRNAs. We selected eleven miRNAs that strongly predicted to target EZH2 and measured their expression. Three miRNAs (miR-26a-5p,miR27a-3p and miR-498) showed significant correlation with EZH2 protein, suggesting them as regulators of EZH2, however miR-26a-5p levels decreased with grade. ChIP analyses revealed H3K27me3 modifications in miR-26a promoter suggesting feedback loop between EZH2 and miR26a. We further measured six downstream miRNA targets of EZH2 and found significant downregulation of four (miR-181a/b and 200b/c) in GBM. Interestingly, EZH2 associated miRNAs were predicted to target 25 genes in glioma-pathway, suggesting their role in tumor formation or progression. Collectively, our work suggests EZH2 and its miRNA interactors may serve as promising biomarkers for progression of astrocytic tumors and may offer novel therapeutic strategies.

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Acknowledgments

We thank Mrs. Kiran Rani and Mr. Pankaj Kumar for their help in immunohistochemistry. We would also like to thank the technical staff of the Neuropathology laboratory, All India Institute of Medical Sciences (AIIMS), New Delhi, India and all the consultants of the Neurosurgery department. This work was funded by the Indian Council of Medical Research (ICMR). Vikas Sharma thanks Council of Scientific and Industrial Research (CSIR) for SRF fellowship. Sonam Takkar thanks Department of Science and Technology (DST-Inspire) for fellowship.

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Authors and Affiliations

  1. Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029, India

    Vikas Sharma, Suvendu Purkait, Prit Benny Malgulwar, Anupam Kumar, Pankaj Pathak, Vaishali Suri, Mehar C. Sharma & Chitra Sarkar

  2. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    Sonam Takkar & Ritu Kulshreshtha

  3. Department of Neurosurgery, All India Institute of Medical Sciences, New Delhi, 110029, India

    Ashish Suri & Shashank Sharad Kale

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  1. Vikas Sharma
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Electronic supplementary material

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Supplementary material 1 (TIFF 705 kb) List of all mRNA and miRNA primers used in quantitative real time PCR studies

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Supplementary material 2 (TIFF 451 kb) EZH2 transcript expression in GBM patient (n=572) and normal brain samples (n=10) based on microarray data from freely available TCGA portal (p-value< 0.0001)

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Supplementary material 3 (TIFF 545 kb) Spearman correlation tables for miRNA targeting EZH2 with EZH2 mRNA and protein levels. The samples with significant correlation (p<0.05 ) have been highlighted

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Supplementary material 4 (TIFF 264 kb) Spearman correlation tables for EZH2 regulated miRNAs with EZH2mRNA and protein levels. The samples with significant correlation (p<0.05) have been highlighted

10014_2015_245_MOESM5_ESM.tif

Supplementary material 5 (TIFF 1398 kb) A diagrammatic representation of the glioma pathway using KEGG program. The genes in the glioma pathway predicted to be targeted by EZH2 associated miRNAs have been highlighted in red

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Sharma, V., Purkait, S., Takkar, S. et al. Analysis of EZH2: micro-RNA network in low and high grade astrocytic tumors. Brain Tumor Pathol 33, 117–128 (2016). https://doi.org/10.1007/s10014-015-0245-1

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  • DOI: https://doi.org/10.1007/s10014-015-0245-1

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