Background
Gliomas are the most common primary intracranial tumor, representing 81% of malignant brain tumors [
1]. According to 2007 World Health Organization (WHO) classification criteria, gliomas can be divided into four grades based on the degree of malignancy (WHO Grade I-IV). WHO Grade I apply to lesions with low proliferative potential and the possibility of cure following surgical resection alone. WHO Grade II and III are called lower-grade gliomas (LGG) [
2]. Because of they are generally infiltrative in nature, surgical resection is not enough, and patients may need adjuvant radiation and/or chemotherapy [
3]. WHO grade IV (reserved for glioblastoma) is the most malignant form of glioma, which has a 5-year relative survival of ~ 5% [
1]. Studies over the past two decades have clarified several genetic alterations in gliomas, such as mutations in
IDH1/2,
TP53 and
ATRX,
TERT promoter mutation,
MGMT promoter methylation and 1p/19q co-deletion, etc. Some of them contribute to glioma classification, prognosis or guidance in therapeutic decisions. In the 2016 WHO classification of central nervous system (CNS) tumors, classification of diffuse gliomas (WHO Grade II-IV) has fundamentally changed: for the first time, a large subset of these tumors is now defined based on
IDH1 or
IDH2 mutation and co-deletion of chromosomal arms 1p and 19q [
4]. This breaks with the principle of diagnosis based entirely on phenotypic by incorporating genotypic parameters into the classification of CNS tumor entities [
4]. The exploit of novel and reliable biomarkers for the prediction of gliomas may further help to elucidate the molecular mechanism of glioma development and progression.
RNA editing is one of the posttranscriptional mechanisms that precisely alters RNA sequences, thus regulates gene expression and generates structurally or functionally different isoforms of proteins. The most predominant pattern of RNA editing converts adenosine to inosine (A-to-I) in coding and non-coding RNA sequences, which is mediated by ADAR enzymes [
5]. A-to-I editing is most abundant in the CNS and is critical for maintaining proper neuronal function [
6]. Targets of this type of RNA editing are transcripts encoding proteins involved in neurotransmitter receptors and voltage-gated ion channels, including α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) [
6], potassium channel Kv1.1 (KCNA1) [
7], G protein-coupled serotonin receptor 5-HT
2CR (5-hydroxytryptamine receptor subtype 2C) [
8] and the α3 subunit of GABA
A (γ-aminobutyric acid type A) receptor (GABRA3) [
9]. Changes in the A-to-I editing have been associated with a number of human diseases, such as amyotrophic lateral sclerosis (ALS), transient forebrain ischemia, epilepsy, metastatic melanoma, glioblastoma (GBM, WHO grade IV) and hepatocellular carcinoma (HCC) [
6,
10,
11].
In human, three ADAR family members (ADAR1, ADAR2 and ADAR3) and two ADAD (adenosine deaminase domain-containing) proteins (ADAD1 and ADAD2) have been identified. They all contain at least one dsRNA binding domain (dsRBD) and a conserved C-terminal deaminase domain, whereas ADAR3 contains a unique Arg-rich ssRNA binding domain (R domain) at its N-terminus [
12]. Unlike the wide expression of ADAR1 and ADAR2, ADAR3 expression is restricted to the brain [
13]. Moreover, ADAR3 is not catalytically active and is thought to act as a competitive inhibitor of ADAR1 and ADAR2 in the brain [
13]. Recently, ADAR3 was proved to directly compete with ADAR2 for binding to
GRIA2 pre-mRNA, inhibiting RNA editing at the Q607R editing site of
GRIA2 in GBM cell line [
14]. This editing site is almost 100% edited in mammalian brain and controls the calcium permeability of AMPA receptor channels, which are involved in fast excitatory synaptic transmission [
15,
16]. These studies suggest that ADAR3 may be associated with the tumorigenesis and progression of glioma. However, the clinical significance and molecular features of glioma with ADAR3 expression remain elusive.
In this study, we evaluated the expression pattern, prognostic significance and potential biological association of ADAR3 in patients with glioma. We collected ADAR3 mRNA expression and clinical information in 1578 glioma samples from four independent datasets. Meanwhile, the expression pattern of ADAR3 in different types of gliomas was evaluated by t test and one-way ANOVA test. In addition, the overall survival of glioma patients was assessed based on ADAR3 expression level and the prognostic value of ADAR3 in glioma was tested using Cox regression analysis. Furthermore, the bioinformatics analyses were applied to predict the biological process of ADAR3 in gliomas. Finally, the relationship of GRIA2Q607R editing and ADAR3 expression has been analyzed based on CGGA RNAseq dataset. These results suggested that ADAR3 was a novel independent prognostic indicator of LGG patients and appear to act as a tumor suppressor in glioma cells.
Discussion
A-to-I editing is the most prevalent transcriptional modification in human cells, which is an endogenous process causing genetic diversity [
5]. In vertebrates, ADAR1, ADAR2 and ADAR3 are main members that catalyzed A-to-I editing [
26]. It has been demonstrated that ADAR enzymes are essential proteins by perturbing expression levels and functions in animal model.
ADAR1 deficient mice die during embryonic development, owing to defective hematopoiesis, widespread apoptosis, liver disintegration and an increasing activity of interferon signaling [
27‐
29].
ADAR2−/− mice became prone to seizures and died within 3 weeks after birth [
30]. The catalytic activity of ADAR3 has not been demonstrated and
ADAR3−/− mice are viable and appear to be normal [
5]. However, the catalytically inactive ADAR3, which localizes exclusively high in the brain, predominantly acts as an inhibitor of editing in the brain through competitive binds to dsRNA substrates [
13,
14,
31].
Furthermore, a majority of transcripts encoding proteins involved in neurotransmission are often targets of A-to-I editing, resulting in changes in the amino acid sequence of protein and physiological function of these ion channels and receptors [
11]. Currently, mutations or changes in expression induced disorder of ADAR activity have been linked to a variety of human diseases, ranging from neurological and neurodegenerative diseases, metabolic diseases, viral infections, autoimmune disorders to cancers [
10]. Previously, ADAR2 has been reported to relate with glioblastoma in both children and adults. ADAR2 promotes
CDC14B editing and overexpression in astrocytoma cells, leading to Skp2 degradation and upregulation of p21 and p27 proteins, consequently causing cells to accumulate in the G1 phase of the cell cycle [
32]. ADAR2 deaminase activity is essential to inhibit glioblastoma proliferation and tumor growth [
32]. Meanwhile, the impaired ADAR2 activity in GBM inhibits a subset of onco-miRNAs (miR221, miR222, miR-21, miR-376a and miR-589–3p) editing, leading to tumor cells proliferation, migration and invasion [
33‐
35]. Recently, Oakes et al. have demonstrated that ADAR3 directly competed with ADAR2 for binding to
GRIA transcript and inhibited RNA editing at the Q/R site of GRIA2 in glioblastoma [
14], and this editing position of GRIA2 was substantially underedited in malignant human brain tumors compared with control tissues [
25]. These suggested that ADAR3 may play a critical role in oncogenesis and development of glioblastoma. However, the clinical and molecular characterizations of ADAR3 in glioma still required further studies.
In this study, we analyzed the expression level of ADAR3 in four independent datasets including 1578 glioma patients. Most notably,
ADAR3 mRNA expression decreased along with WHO grade progression, suggesting that the expression of
ADAR3 gradually attenuated with the malignant increase of pathological glioma. Moreover, the
ADAR3 expression level was significantly highest in the phenotype of known favorable molecular, such as neural subtype and LGG IDH-mut and 1p/19q codeleted stratified patients. The association of ADAR3 with glioma progress indicates a tumor suppressor role of ADAR3 in the tumorigenesis and progression of glioma. However, an increase in ADAR3 protein expression in the tumor tissue compared to adjacent tissue was observed in 5 out of 6 glioblastoma patient samples in previous study [
14]. Thus, inconsistencies between different studies suggest the possibility that ADAR3 protein expression or activity may be modulated by a post-transcriptional way. At the same time, further study will be needed to investigate ADAR3 protein expression based on a large cohort.
From the overall survival curve, the low expression of
ADAR3 indicated shorter overall survival time and lower survival rate in glioma patients. After divided the patients into LGG and GBM subgroup, the high expression of
ADAR3 is also a favorable indicator in LGG group, but not in GBM group. Furthermore, the high expression of ADAR3 was associated with longer overall survival time in LGG IDH-mut and 1p/19q non-codeleted and LGG IDH-wt patients. Combined with univariate and multivariate Cox analysis,
ADAR3 expression is an independent prognostic factor in patients with diffuse glioma, especially in LGG group. Recently, several genes have been reported to be prognostic factors in gliomas, such as IDH1 [
36], FGFR3 [
37], Notch1 [
38]. Our research illuminated the clinical features of ADAR3 in diffuse glioma and confirmed that
ADAR3 expression had a guiding significance for the prognosis of patients with LGG.
Through an analysis of the biological process of ADAR3 in glioma, we found that ADAR3 play an important role in the normal biological process, such as signal transduction, chemical synaptic transmission, neurotransmitter transport and intracellular signal transduction. This is consistent with A-to-I editing targets, which are primarily transcripts of neurotransmitter receptors and ion channels proteins [
11]. Meanwhile, downregulation of ADAR3 will promote cell proliferation, angiogenesis, cell adhesion and migration. This indicated that inhibition the expression of ADAR3 in brain cells would promote the cell malignant transition. Collectively, these data strongly support the tumor suppressor role of ADAR3 in glioma progression.
The reduced GRIA2 editing at Q607R site has been observed in malignant gliomas [
25], and the unedited GRIA2 protein promotes cell migration and invasion in these cell lines [
24]. Oakes et al. reported that overexpression of ADAR3 inhibited RNA editing at the Q607R site of GRIA2 in astrocyte and astrocytoma cell lines [
14], which indicted the competitive inhibition of ADAR2 with ADAR3 on this site. Based on our clinical data, we found the editing level of GRIA2
Q607R is positively related with ADAR2 and ADAR3 mRNA expression, which is inconsistent with previous in vitro assay. These results indicated that the regulation of GRIA2 editing in gliomas is a more complex model than previous studies, and the tumor suppressor role of ADAR3 may partly related with the underedited level of Q607R.
Authors’ contributions
YZ and KYW provided equal contributions to the design of the study, data analysis. ZZ, SS, KNZ, RYH and FZ participated in data downloading and preliminary analysis. YZ planned and wrote the manuscript. HMH critically revised the manuscript. All authors have read and approved the final manuscript.