Background
Long non-coding RNAs (lncRNA) were a group of noncoding RNAs with more than 200 bp, which has intrinsic advantages over the use of protein-coding RNAs in diagnostics [
1]. LncRNAs play critical roles in diverse biological functions, including nuclear architecture, regulation of gene expression, immune surveillance, cancer development and maintenance of tumorigenesis. LncRNAs can regulate miRNAs and mRNAs by sequestering and binding them, and competing endogenous RNA (ceRNA) mechanism attracted more and more attention since it was firstly proposed by Salmena et al. [
2]. CeRNA is a complex post-transcriptional regulatory network using miRNA response elements (MREs) to compete for the binding of miRNAs thereby implementing mutual control between mRNAs, lncRNAs and miRNA [
3]. A lot of studies have reported that ceRNA mechanism plays critical roles in solid tumor progression [
4‐
6]. Junge et al. [
7], Ding et al. [
8] and Chen et al. [
9] also reported highly-upregulated RUNX1T1, C-Myc and CCAT1 acts as competing endogenous RNA (ceRNA) in adult acute myeloid leukemia. However, little has been done in pediatric and adolescent cytogenetically normal acute myeloid leukemia (CN-AML) patients. Although board overlap lies between AML adults and pediatric patients in pathogenesis, diagnosis, treatment and prognosis of the diseases, differences still exist.
CN-AML, a most common AML type, is characterized by the absence of microscopically detectable chromosome abnormalities, but mutations, epigenetic changes and dysregulated expression signatures exists, respectively [
10]. Some reports have shown CN-AML patients harboring mutations including NPM1, CEBPA, FLT3-ITD and WT1 were associated with an disparate prognosis [
11]. With the advent of high-throughput technologies such as microarray and next-generation sequencing, some prognostic gene expression signatures have been proposed. These findings helped focus targeted therapies for CN-AML with significantly heterogeneous outcomes [
12]. The genes in a prognostic gene signature were considered as independent individuals, which may result in ignoring potential relationships between the genes [
13]. To overcome these challenges and to reduce the difficulty in biological interpretation, we constructed a weighted correlation network and the significant modules associated with prognosis were selected [
14]. Based on these modules information, we identified 4 biological pathways associated with overall survival (OS). Next the most enriched pathway was selected to verify whether it could accurately predict pediatric and adolescent CN-AML patients’ prognosis and guide treatment. To our knowledge, our study is the first to construct survival specific ceRNAs co-expression network and identify survival specific biological pathways in pediatric and adolescent CN-AML patients. This approach can help to clarify the functions of lncRNAs in younger CN-AML patients.
Discussion
Pediatric and adolescent acute myeloid leukemia (AML) with incidence of approximately 7 occurrences per 1 million children annually is a rare type of childhood cancer [
19]. Because high incidence of severe and dose-limiting short- and long-term toxicities happened, younger AML therapy was a big challenge for patients, their families, and care providers [
20]. Although multiple national and international cooperative groups have contributed to an evolving treatment strategy including four to five kinds of intensive myelosuppressive chemotherapy and stem cell transplantation over several decades, there was only a mild decline in younger AML mortality. Do¨hner et al. [
21] has been reported State-of-the-art recommendations in adult AML. But little has been done in pediatric and adolescent AML patients. Although there were broad overlap in diagnosis, treatment and prognosis for AML, differences still exist between adolescent and adult patients [
11]. Hence, to improve younger AML’s prognosis, the pivotal genes and regulatory mechanism in AML’s development and progress need to be identified. Studies revealed that lncRNAs play important roles in diverse gene expression and cellular processes regulation. Dysregulation of lncRNAs has been reported to contribute to oncogenesis and tumor metastasis, including AML [
22,
23].Therefore, the investigation of lncRNAs’ expression and function could help us to understand leukemogenesis and identify novel therapeutic targets. However, to date, only a few studies have reported mechanical and functional characterization of AML-associated aberrant gene networks [
24‐
27].
Recently, many studies have reported that endogenous lncRNA had miRNA responsive elements (MRE) and modulated miRNAs via acting as miRNA sponges and binding with them [
28]. These lncRNAs acting as competing endogenous RNAs (ceRNAs) participate in post-transcriptional regulation by interfering with the miRNA pathways [
29]. ceRNAs have been shown to play critical roles in diverse biological functions and the disruption of the equilibrium of ceRNAs were implicated in tumorigenesis [
3,
28]. For example, ATB lncRNA blocked miR-200 family by binding to its targets and upregulated ZEB1 and ZEB2, thereby inducing EMT and invasion in hepatocellular carcinoma [
30]. Hence, understanding the intricate interplay among protein-coding messenger RNAs, miRNAs and lncRNA would help to identify gene regulatory networks which played critical roles in the progress and development of younger CN-AML. In the present study, we identified prognosis related specific lncRNAs, miRNAs and mRNAs of CN-AML from TCGA database. Moreover, we constructed a ceRNA network which could provide an integrated biological view based on the bioinformatics differential analysis. Some of these network have been reported to be solid tumor-associated gene-network, such as lncRNA–miRNA (CASC2-miR-221 [
31]), miRNA–mRNA (miR-221-RAB1A [
32] ,miR-25-PTEN [
33], and miR-221-FMR1 [
34]). The ceRNA network that we built revealed a previously unknown ceRNA regulatory network in pediatric and adolescent CN-AML. However, we failed to find target mRNAs when we constructed a ceRNA network with lncRNAs positively associated with overall survival and miRNAs negatively associated with prognosis.
In order to identify the function of the key lncRNAs, miRNAs and mRNAs in the ceRNA network, we analyzed their associations with OS by Kaplan–Meier curve. Our results suggested that 6 lncRNAs, 1 miRNA and 14 mRNAs were associated with CN-AML overall survival. Among them, has-mir-363 has been reported to be associated with prognosis of AML [
35]; lncRNA CRNDE played critical roles in promoting cell proliferation, invasion and migration of solid tumor [
36]; mRNA HMBOX1 and KIF5B was involved in the carcinogenesis [
37,
38]. The other lncRNAs (AL356475.1, AC011498.1, AC092811.1, LINC00158 and LINC00504) and mRNAs (BRWD1, CNIH1, FMR1, GOLGA8J, GPBP1L1, NUFIP2, PTAR1, RRN3, SNX4, TMF1, ZFC3H1 and ZKSCAN8) were not reported previously. We analyzed the relationships of the above 35 key lncRNAs, 7 miRNAs with clinical features including age at diagnosis, gender, WBC at diagnosis, bone marrow blasts, peripheral blasts and molecular mutations (FLT3-ITD, NPM1, CEBPA). The results showed that AC011498.1, CRNED, LINC00504 and hsa-mir-363 were the indicators of CEBPA mutation of AML. Furthermore, AC011498.1 was also associated with NPM1 mutation. LncRNA CRNDE and miRNA hsa-mir-363 have been previously reported to be associated with clinical features of leukemia [
35,
39]. However, the other lncRNAs we found here have not been reported to be the indicators of relevant features previously. The imprint of genetic ancestry and population structure carried in the genome of each individual and groups has led to the remarkable racial and ethnic diversity, which integrate biological, geopolitical, linguistic and cultural factors and are widely applied in population study [
40]. Besides, Bonham et al. [
41] have reported that the assessment of race and ethnicity at the individual level will make us closer to more individualized genetic-based medicine. Hence, we have estimated the association between miRNAs, race and ethnicity, but we failed to find any relation among them.
To date, the prognostic gene expression signatures have been extensively proposed for targeted therapies in cancer patients with significantly heterogeneous outcomes. However, the potential relationships of the genes have been ignored [
13]. Genes were considered as independent individuals, which may result in undermining potential relationship between genes [
13]. To overcome these challenges and to reduce the difficulty in biological interpretation, we constructed a weighted correlation network [
14]. Then, we analyzed the relationships among the modules, OS, FAB category and the molecular mutations associated with prognosis (FLT3-ITD, NPM1, CEBPA). To identify biological pathways that were indicators of AML’s prognosis, significant modules associated with OS and clinical features were selected to carry out functional enrichment analysis. The GO analysis showed that the functions of significant differences in the aspects of “transcription, DNA-templated” (ontology: BP), “integral component of membrane” (ontology: CC) and “DNA binding” (ontology: MF). Furthermore, by KEGG pathway analysis, we identified 4 biological pathways associated with OS which may predict prognosis and guide treatment in younger CN-AML patients, including ‘Transcriptional misregulation in cancer, Regulation of actin cytoskeleton, TGF-beta signaling pathway and Ether lipid metabolism’. Recently, many studies have shown epigenetic abnormalities caused by ‘Transcriptional misregulation in cancer’ play important roles in tumor biology, such as DNA methylation, histone modifications and noncoding RNAs [
42‐
44]. Therefore, we selected the most enriched network ‘Transcriptional misregulation in cancer’ to verify the relationship between epigenetic abnormalities and CN-AML patients’ prognosis. The result showed that the ‘Transcriptional misregulation in cancer’ pathway could strongly predict younger CN-AML patients’ survival. In our study, we only showed FLT3-ITD mutations could predict unfavorable outcome of pediatric CN-AML patients (P = 0.003), but we failed to find the relationship between NPM1 and CEBPA mutation and patients’ prognosis. One reason may be the sample size in our study was small and it couldn’t come to a significant conclusion. Another reason may be that patients simultaneously possess FLT3-ITD, NPM1 or CEBPA mutations in our study. Some reports have shown FLT3-ITD could implement a negative effect on OS irrespective of NPM1 or CEBPA mutations [
45‐
47].
In PPI network analysis, we identified 30 hub genes and selected the top ten genes to analysis in detail. Among them, the PIK3CG gene, which encodes the catalytic subunit of phosphoinositide 3-OH-kinase-γ (PI3Kγ) namely p110γ, is located in chromosome band 7q22 and is often missing in myeloid malignancies [
48]. Grimwade et al. [
48] has implied PIK3CG was evaluated as a candidate suppressor gene of myeloid tumor. Dysregulation of apoptosis, which leads to the accumulation of tumor cells by slowing the rate of cell turnover, is a hallmark of cancer [
49]. BCL-2 proteins encoded by BCL-2 gene can be classified into two families, anti-apoptotic and pro-apoptotic proteins. As a central regulator of cell polarity, loss of CDC42 suppresses AML cell polarity and division asymmetry [
50]. Primary AML cells show constitutive release of a wide range of chemokines (including CCL5) involved in leucocyte chemotaxis, differentiation and angiogenesis [
51]. LEP acting as a growth factor promotes cellular proliferation and may affect leukemic hematopoiesis [
52]. Hence, the genes PIK3CG, BCL-2, CDC42, CCL5 and LEP in the treatment of AML are now being widely and actively taken into consideration. In previous studies, there were no reports showing the other top ten hub genes were associated with AML.
Authors’ contributions
XY collected and analyzed the data, wrote the paper; SH research literature and edit the paper; FJF and RZ revise the paper; CYS and YH conceived and designed this study, analyzed the data, wrote the paper; and all authors reviewed the paper, and approved the final manuscript. All authors read and approved the final manuscript.