International Journal of Molecular Sciences Article The Long Non-Coding RNA H19 Drives the Proliferation of Diffuse Intrinsic Pontine Glioma with H3K27 Mutation David Roig-Carles 1, Holly Jackson 1, Katie F. Loveson 2 , Alan Mackay 3, Rebecca L. Mather 1, Ella Waters 1, Massimiliano Manzo 4, Ilaria Alborelli 4 , Jon Golding 1 , Chris Jones 3, Helen L. Fillmore 2 and Francesco Crea 1,* 1 Cancer Research Group, School of Life, Health and Chemical Sciences, The Open University, Milton Keynes MK7 6AA, UK; [email protected] (D.R.-C.); [email protected] (H.J.); [email protected] (R.L.M.); [email protected] (E.W.); [email protected] (J.G.) 2 School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2UP, UK; [email protected] (K.F.L.); helen.fi[email protected] (H.L.F.) 3 Division of Molecular Pathology, The Institute of Cancer Research, London SW7 3RP, UK; [email protected] (A.M.); [email protected] (C.J.) 4 Institute of Pathology, University Hospital Basel, 4031 Basel, Switzerland; [email protected] (M.M.); [email protected] (I.A.) * Correspondence: [email protected] Abstract: Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identi- fying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions Citation: Roig-Carles, D.; Jackson, have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development- H.; Loveson, K.F.; Mackay, A.; Mather, associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure R.L.; Waters, E.; Manzo, M.; Alborelli, the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and I.; Golding, J.; Jones, C.; et al. The H19 Long Non-Coding RNA H19 Drives sub-cellular location of lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense the Proliferation of Diffuse Intrinsic oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expres- Pontine Glioma with H3K27 sion was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in Mutation. Int. J. Mol. Sci. 2021, 22, decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, 9165. https://doi.org/10.3390/ resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first ijms22179165 functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer. Academic Editor: Athanasios G. Papavassiliou Keywords: paediatric glioma; DIPG; diffuse midline glioma; lncRNA; epigenetics; brain cancer Received: 16 July 2021 Accepted: 19 August 2021 Published: 25 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral Malignant brain tumours are a leading cause of death in paediatric patients. Diffuse with regard to jurisdictional claims in intrinsic pontine glioma (DIPG) is a type of paediatric high-grade glioma (pedHGG) published maps and institutional affil- originating in the brainstem and affecting children with a median age of six to seven iations. years [1,2]. DIPGs are highly infiltrative and belong to the fibrillary astrocytoma family, where lesions are classified as either WHO grade III or IV [3]. At the molecular level, substitution of lysine at the 27 position of the histone 3 locus with methionine (H3K27M at either H3.1 or H3.3) has been suggested to drive the oncogenesis of DIPGs [4]. H3K27M substitution is identified in approximately 80% of histologically confirmed DIPGs. The Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. World Health Organization has recently classified DIPGs in the broader category of diffuse This article is an open access article midline gliomas (DMGs) with a H3K27M mutation [2,5–7]. Another frequent mutation of distributed under the terms and the H3 locus, GR4R, is more prevalent in pedHGGs located in the cerebral hemispheres [8]. conditions of the Creative Commons The surgical removal of DIPGs is almost impossible due to their infiltrative nature and their Attribution (CC BY) license (https:// location. Currently, radiotherapy is the standard treatment for this malignancy; despite this creativecommons.org/licenses/by/ treatment, the median survival for DIPG patients is 9–11 months [9,10]. Therefore, novel 4.0/). therapeutic targets are necessary to improve the prognosis of these paediatric patients. Int. J. Mol. Sci. 2021, 22, 9165. https://doi.org/10.3390/ijms22179165 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2021, 22, 9165 2 of 13 There are approximately 60,000 long non-coding RNAs (lncRNAs) in the human ge- nome; these transcripts are defined as non-coding transcripts longer than 200 nucleotides. SomeThere lncRNAs are approximately are highly expressed 60,000 longunder non-coding pathological RNAs conditions (lncRNAs) and regulate in the human key as- genome;pects of thesetumour transcripts progression, are defined invasion, as non-codingand metastasis transcripts [11,12]. longerPrevious than studies 200 nucleotides. have iden- Sometified lncRNAs several lncRNAs are highly associated expressed with under DIPG pathological progression conditions [13,14]. and However, regulate no key lncRNAs aspects ofhave tumour been progression, functionally invasion,characterised and metastasisin this malignancy. [11,12]. Previous studies have identified severalH19 lncRNAs (gene ID: associated 283120) is with an extensively DIPG progression studied [ 13lncRNA,,14]. However, whose aberrant no lncRNAs expression have beenhas been functionally linked to characterised alterations during in this malignancy.foetal development [15]. The oncogenic role of H19 has H19been(gene described ID: 283120) in several is an malignancies, extensively studiedincluding lncRNA, adult gliomas whose aberrant[16–18]. Mechanisti- expression hascally, been H19 linked expression to alterations is triggered during by foetal hypoxia; development this lncRNA [15]. can The affect oncogenic a wide role range of H19 of hashub beengenes, described including in severalmicroRNAs malignancies, (miRNAs) including and mRNAs adult [19,20]. gliomas However, [16–18]. Mechanistically, the function of H19H19expression in pedHGG is triggeredstill remains by hypoxia;largely unknown. this lncRNA can affect a wide range of hub genes, includingHerein, microRNAs we investigated (miRNAs) the andclinical mRNAs relevance [19,20 of]. H19 However, in DIPG the cells function and tested of H19 H19 in pedHGGtargeting still for remainshalting DIPG largely proliferation. unknown. Herein, we investigated the clinical relevance of H19 in DIPG cells and tested H19 targeting2. Results for halting DIPG proliferation. 2.1. H19 Is Up-Regulated in DIPG Tissue 2. Results 2.1. H19To Isconfirm Up-Regulated the expression in DIPG of Tissue H19 lncRNA in DIPG tissue, we analysed open-access clinical datasets of pedHGGs and normal brain tissue. Bioinformatic analyses confirmed To confirm the expression of H19 lncRNA in DIPG tissue, we analysed open-access that H19 levels are significantly increased in the DIPG tissue of the “Allis-45-custom-ilm- clinical datasets of pedHGGs and normal brain tissue. Bioinformatic analyses confirmed nht12v4” dataset (p < 0.01, Figure 1a) whereas an increasing trend was observed in that H19 levels are significantly increased in the DIPG tissue of the “Allis-45-custom- ilmnht12v4”“Paugh-37-MAS5.0-u133p2” dataset (p < 0.01, (Figure Figure 11b).a) whereas Microarray an increasing data from trend Paediatric was observed Cbioportal in “Paugh-37-MAS5.0-u133p2”(https://pedcbioportal.kidsfirstdrc.org/, (Figure1b). access Microarrayed 30 June data 2020) from demonstrated Paediatric Cbioportalsignificantly (https://pedcbioportal.kidsfirstdrc.org/higher H19 expression in the brainstem than, accessed in hemispheric 30 June 2020) pedHGGs demonstrated (p < 0.01). signifi-(Figure cantly1c). We higher then H19exploredexpression the influence in the brainstemof mutations than in in the hemispheric H3 genes (H3K27M pedHGGs and (p <WT) 0.01). on (FigureH19 expression;1c). We then this explored analysis the revealed influence that of mutationsH3K27M-bearing in the H3 pedHGGsgenes (H3K27M expressand higher WT) onlevelsH19 ofexpression; H19 compared this analysis to pedHGGs revealed bearing that H3K27M-bearingthe wild-type H3 gene pedHGGs (p < 0.01) express (Figure higher 1d). levelsHistological of H19 comparedclassification to pedHGGsof pedHGG bearing samples the showed wild-type thatH3 DIPGgene tissues (p < 0.01) express(Figure higher1d) . HistologicalH19 levels than classification other pedHGGs, of pedHGG including samples anaplastic showed astrocytoma that DIPG tissues and glioblastoma express higher (p < H190.01)levels (Figure than 1e). otherOverall pedHGGs, survival analysis including did anaplasticnot identify astrocytoma significant differences and glioblastoma between (phigh-< 0.01) and (Figure low-H191e).-expressing Overall survival DIPG groups analysis in didthe notCBioportal identify dataset significant (median differences survival betweenwas 9.8 and high- 11.1 and months, low-H19 respectively)-expressing (Figure DIPG groups1f). Analysis in the of CBioportal a single-cell dataset RNA (mediansequenc- survivaling dataset was from 9.8 and the11.1 developing months, human respectively) midbrain