The Dynamic m⁶A Epitranscriptome in Glioma Stem Cell Plasticity and Function

Glioblastoma multiforme is one of the most aggressive tumors of the central nervous system. The current standard-of-care includes maximal resection followed by chemotherapy, radiation and more recently, tumor treating fields (TTFs). Despite this multimodal approach, glioblastoma remains refractory to therapy. Glioblastoma resistance, recurrence and malignancy are believed to be driven by a subpopulation of glioma stem cells (GSCs) within the tumor bulk which are characterized by the retention of self-renewal potential as well as the capacity to recapitulate tumor heterogeneity. Within the dynamic intratumoral niche, GSCs demonstrate a high degree of cellular plasticity, reversibly interconverting between stem-like states and more differentiated states as a result of environmental cues/signaling fluctuations. Such plastic adaptive properties are mostly driven by multiple dynamic, reversible epigenetic modifications. We posit that reversible post-transcriptional methylation of RNA transcripts at the m6A position may be one such regulatory mechanism employed by GSCs to efficiently maintain plasticity and adaptive phenotypic transitions. In this section, we discuss the concept of cellular plasticity, introduce dynamic m6a epitranscriptomic mechanisms as potential key regulators of GSC plasticity and finally propose epigenetic based therapeutics as a mean of attenuating glioblastoma plasticity to improve patient outcome

Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following <em>in</em> <em>vivo</em> PNS Injury

<div><p>Injury response in the peripheral nervous system (PNS) is characterized by rapid alterations in the genetic program of Schwann cells. However, the epigenetic mechanisms modulating these changes remain elusive. Here we show that sciatic nerve injury in mice induces a cohort of 22 miRNAs, which coordinate Schwann cell differentiation and dedifferentiation through a combinatorial modulation of their positive and negative gene regulators. These miRNAs and their targeted mRNAs form functional complexes with the Argonaute-2 protein to mediate post-transcriptional gene silencing. MiR-138 and miR-709 show the highest affinity amongst the cohort, for binding and regulation of Egr2, Sox-2 and c-Jun expression following injury. Moreover, miR-709 participates in the formation of epigenetic silencing complexes with H3K27me3 and Argonaute-1 to induce transcriptional gene silencing of the Egr2 promoter. Collectively, we identified a discrete cohort of miRNAs as the central epigenetic regulators of the transition between differentiation and dedifferentiation during the acute phase of PNS injury.</p> </div

miRNAs mediate transcriptional gene silencing of Egr2.

<p>(A). Real-time qRT-PCR of Egr2 transcripts normalized to GAPDH control 6, 24 and 48 hours post-injury as compared to control uninjured nerves. Fold difference (2^-ΔΔCT) between injured and control nerves is plotted and error is expressed as standard deviation. Inset: Egr2 mRNA expression at 24 hours and 48 hours after injury as compared to Egr2 mRNA expression in uninjured nerves. Egr2 mRNA expression is completely inhibited 48 hours post-injury. Beta-actin was used to show equal loading and amplification. (B). Nuclear “run-on” experiment for nascent Egr2 mRNA transcription in rat Schwann cells transfected with control or miR-709 duplexes as measured by quantitative RT-PCR and normalized to GAPDH mRNA transcription levels. The experiment was repeated three times, the data were normalized to GAPDH and fold difference (2^-ΔΔCT), between injured and control nerves was plotted as a log-2 median ratio. Error is expressed as standard deviation, (**: p<0.005). (C). Egr2 protein expression in Schwann cells transfected with miR-709, antimiR-138 or non-targeting control miRNA and compared with non-transfected Schwann cells. (D). Methylation PCR analysis (SA Biosciences) of the CpG islands of the proximal Egr-2 promoter from control and injured sciatic nerves (24h-post injury). The relative percentages of hypermethylated (HM) and unmethylated (UM) fractions were calculated by comparing the amount in each digest with that of a mock (no enzyme added) digestion. (E). ChIP assays, using antibody to H3K27Me3 or no antibody (No Ab) controls were performed in sciatic nerves isolated from control and axotomized mice 48 hours post-injury. Western blot of input, no antibody control (No Ab) and H3K27Me3-ChIP was performed with antibodies against Ago-1 and H3K27Me3. This showed that Ago-1 is enriched in H3K27me3 silencing complexes following <i>in vivo</i> peripheral nerve injury. (F). Real-time quantitative PCR to assess the presence of the MSE region of the Egr2 promoter (left bars) and miR-709 (right bars) in the H3K27me3 immunoprecipitated chromatin. Lysates pre-treated with RNase H were immunoprecipitated to confirm RNA-DNA interaction. Values for the MSE and miR-709 were normalized to input DNA and miRNA respectively. The fold difference (2^-ΔΔCT) in the association of MSE and miR-709 with H3K27Me3 complex between injured and control nerves, was plotted as a log-2 median ratio and the error is expressed as standard deviation. (**: p<0.001). Note the significant increase in the association of miR-709 and MSE with H3K27me3 silencing complexes following <i>in vivo</i> nerve injury.</p

Protein expression changes and miRNA expression profile following <i>in vivo</i> sciatic nerve injury.

<p>Protein expression analysis reveals a robust reduction in pro-myelination and upregulation of anti-myelination factors. All experiments were repeated three times unless otherwise stated. (A). Uninjured and distal segments of axotomized sciatic nerves at 48 hours post injury (PI) (n = 4) were lysed in SDS buffer and western blots were performed for Egr2 (63 kDa), C-Jun (43 kDa), Sox-2 (35 kDa), ID-2 (15 kDa), Nanog (34–40 kDa), QKI-6 (38 kDa), P75^NTR (75 kDa), beta-actin (42 kDa) and Histone H3 (17 KDa). (B). Relative protein levels normalized to beta-actin were plotted to demonstrate the differential expression of the pro-myelination and anti-myelination factors in injury. Statistical significance was calculated with a Student’s t-test (**: p<0.001). (C). Microarray for miRNAs (version 9.2) was performed using total RNA from 15 control and 15 axotomized mouse sciatic nerves at 6 hours and 24 hours post-injury (PI), by Exiqon (Vedbaek, Denmark). The array was repeated twice and Log2 median ratios of the miRNAs expressed in 24h-injured nerve compared to the control were plotted and the standard error was calculated.</p

Autotransfecting Short Interfering RNA through Facile Covalent Polymer Escorts

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes

miRNA-mediated loss of m6A increases nascent translation in glioblastoma.

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition