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Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

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Abstract

Introduction

Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression.

Results

In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression.

Conclusions

These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

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Acknowledgments

We are grateful and wish to extend our thanks to Dr Kim Haworth for assistance with pyrosequencing. This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Conflict of interest

We hereby confirm that there is no financial or personal relationship between the authors and other people or organizations that can inappropriately influence the work and there is thereby no conflict of interest.

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Authors and Affiliations

  1. Human Disease and Genomics Group, Institute of Science and Technology in Medicine, School of Medicine, Keele University, Stoke-on-Trent, Staffordshire, ST4 7QB, UK

    Mark O. Kitchen, Kiren Yacqub-Usman, Alan Richardson, Richard N. Clayton & William E. Farrell

  2. School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, College Road, Sutton Bonington, Leicestershire, LE12 5RD, UK

    Richard D. Emes

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  1. Mark O. Kitchen
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  2. Kiren Yacqub-Usman
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Correspondence to William E. Farrell.

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Mark O. Kitchen and Kiren Yacqub-Usman have contributed equally to this work.

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11102_2014_630_MOESM3_ESM.pdf

Expression and histone tail modifications associated with HMGA-targeting miRNA in human pituitary adenomas. (A) From left to right, quantitative RT-PCR of miR-16, miR34b and miR-320 in non-functioning (NF), prolactinoma (P), corticotrophinoma (A) and growth hormone secreting adenomas (GH). Expression is reported relative to the mean of three post-mortem pituitaries, where the mean value was expressed as equal to 1. Each bar represents the mean value ± SEM from three independent experiments performed in triplicate. (B) Chromatin immunoprecipitation analysis (ChIP) for the histone tail modification associated with active genes, H3K9Ac. Adenomas, and three post-mortem normal pituitaries, are those shown in panel A and enrichment in each case is relative to input chromatin. (C) ChIP analysis of adenomas and post-mortem normal pituitaries shown in panels A and B, for the histone tail modification associated with silenced gene, H3K27Me3. In panels B and C each bar represents mean value ± SEM from three independent experiments performed in triplicate

11102_2014_630_MOESM4_ESM.pdf

Epidrug mediated effects on hmga1 targeting miRNA (A) From left to right, expression of miR-16, miR-34b and miR-320 in GH3 cells as determined by RT-qPCR and relative to normal rat pituitaries (NRP). Expression was determined in the absence or presence of the epidrugs, zebularine (Zeb) and trichostatin A (TSA) either alone or in combination. Doses of drugs are shown on the x axis. Expression is reported relative to the mean of three normal rat pituitaries (NRP), where the mean value was expressed as equal to 100 % and where each bar represents the mean value ± SEM from three independent experiments performed in triplicate. Panels B and C show the ChIP analysis of the cells shown in panel A, where panel B is the modification associated with active gene, H3K9Ac and panel C is the modification associated with silencing, H3K27me3. Each bar represents the mean value ± SEM from three independent experiments performed in triplicate. Data was analyzed for significance by one-way ANOVA with Dunnett’s multiple comparison post-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus NRP

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Kitchen, M.O., Yacqub-Usman, K., Emes, R.D. et al. Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells. Pituitary 18, 674–684 (2015). https://doi.org/10.1007/s11102-014-0630-5

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