Competitive Endogenous RNA (ceRNA) Regulation Network of lncRNA–miRNA–mRNA in Wilms tumor CURRENT STATUS:

Background: Competitive endogenous RNA (ceRNA) have revealed a new mechanism of interaction between RNAs. However, such comprehension of the ceRNA regulatory network in wilms tumor remains limited. Methods: Raw RNA sequencing profiles regarding mRNAs, miRNAs and lncRNAs on wilms tumor samples and normal samples were obtained from Therapeutically Applicable Research to Generate Effective Treatment (TARGET). EdgeR package was applied to identify differentially expressed lncRNAs, miRNAs and mRNAs. Functional enrichment analyses were conducted via DAVID database and the ClusterProfile R package. The lncRNA–miRNA–mRNA interaction ceRNA network was established in Cytoscape according to the identified lncRNAs–miRNAs and miRNAs–mRNAs interactions. Subsequently, correlation between ceRNA network and overall survival prognosis were analyzed. Results: A total of 2,037 lncRNAs, 154 miRNAs and 3,609 mRNAs were identified as differentially expressed RNAs in wilms tumor. 205 lncRNAs, 26 miRNAs and 143 mRNAs were included in ceRNA regulatory network. Analysis results showed that 14 out of the 205 lncRNAs, 1 out of 26 miRNAs and 8 out of 143 mRNAs were associated with overall survival in Wilms tumor patients (P < 0.05). Conclusions: CeRNA networks played an important role in Wilms tumor. This might provide effective bioinformatics basis and novel insights for further understanding of the mechanisms underlying Wilms tumor.


Background
Wilms tumor (WT) is the most common type of pediatric renal malignancy. WT have a poor prognosis although 5-year overall survival rate is constantly improved with the advancement of disease-associated therapies [1]. Chemotherapy, surgery and radiation therapy are the main treatment strategies for WT. However, 50% of children who have a recurrence after these treatments go on to die from this tumor [2,3]. Novel therapeutic 3 treatment targeting specific mechanisms of WT is still lacking.
Previous studies demonstrated that that numerous key Long non-coding RNA (lncRNA), microRNA (miRNA) and mRNAs are closely related with pathogenesis of WT, such as LINC00473 [4], miR-483-5p [5], miR-195 [4] and HACE1 [6]. However, there were little reports in prognosis biomarkers in WT. If WT patients who were more likely to have a poor prognosis according to these prognosis biomarker result could be identified, clinician might apply more aggressive treatment. if WT patients with low risk could be identified, treatment with less morbidity could be administered. Prognosis biomarkers and targeted cure in WT were required to be identified in order to improve the clinical outcomes.
In the last decade, complexity of the human genome could be revealed by advanced RNA sequencing analysis [7]. Under such circumstance, competing endogenous RNA (ceRNA) hypothesis was presented which demonstrated that lncRNAs could communicate with common miRNA response elements with miRNA to construct anintricate interplay network and ultimately crosstalk with mRNA. The involvement of ceRNA regulatory network tumor initiation and progression was validated in previous studies [8,9]. However, specific ceRNA regulatory network (lncRNA-miRNA-mRNA) in WT still remained unelucidated.
In present study, ceRNA regulatory network (205 mRNAs, 26 lncRNAs and 143 miRNAs) was constructed to promote the understanding how lncRNAs sponged miRNA to regulated gene expression in WT. Subsequently, survival analysis and functional analysis were used to promote new understanding of the role of ceRNA regulatory network in WT carcinogenesis. The present study might give insight into molecular mechanism which participate in the progression and tumorigenesis of WT.

Data collection and preprocessing
Data were retrieved from Therapeutically Applicable Research to Generate Effective 4 Treatment (TARGET:https://ocg.cancer.gov/programs/target) database. These data (of lncRNAs, mRNAs and miRNAs) in present study were publicly available. Ethics committee approval was not required because data in present study was obtained from TARGET database. Among them, miRNA expression data were acquired from 138 samples, including 6 normal samples and 132 WT samples. And mRNA and lncRNA expression data were selected from 132 samples, including 6 normal samples and 126 WT samples. The differentially expressed lncRNAs (DELs), differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between WT and normal samples were carried out via EdgeR package in R software (Version 3.2.2). |logFC(fold change)| >1 and false discovery rate (FDR) of <0.05 were defined as cut-off criteria.

CeRNA network construction and functional enrichment analysis
Cytoscape software (Version 3.6.1) was utilized to construct and visualize the DELs-DEMs-DEGs ceRNA network. Cytoscape was an effective software to visualize the molecular interaction networks according to gene expression profiles and annotations. In order to better comprehend the tumorigenesis mechanisms in WT, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) of DEGs in ceRNA network were performed via DAVID (the Database for Annotation, Visualization and Integrated Discovery). The limit of value is that FDR <0.05.

Survival analysis
Identification of prognostic DELs, DEMs and DEGs signatures were executed via the logrank test and Kaplan-Meier analysis. The survival curves were constructed using the 'survival' package. Survival analysis was performed according to Kaplan-Meier univariate survival analysis. P<0.05 were selected as statistical threshold value. All the survival analysis was conducted in R software (version: 3.3.2).

DELs, DEGs and DEMs in WT.
LncRNA, mRNA, and miRNA expression profiles between WT samples and normal samples acquired from TARGET were analyzed in present study. 2,037 DELs, 154 DEMs and 3,609 DEGs, were screened out in present study. 1,247 up-regulated and 790 down-regulated DELs were identified in WT with cut-off threshold of |logFC(fold change)| >1 and false discovery rate (FDR) of <0.05. DELs distribution between WT and normal controls were presented as heatmap plot in Figure 1A. According to the cut-off threshold of |logFC(fold change)| >1 and false discovery rate (FDR) of <0.05, 105 up-regulated and 49 downregulated DEMs were identified in WT. Heatmap plot of the related DEMs between WT and normal controls is shown in Figure 1B. A total of 1,894 up-regulated and 1,715 downregulated DEGs were identified in WT. DEGs distribution between WT and normal controls were presented as a heatmap plot in Figure 1C. The top 10 upregulated and downregulated DELs, DEMs and DEGs were showed in Table 1. GO and KEGG analysis were also performed to reveal the functions of the 16 DEGs which were included in the ceRNA network. For "biological processes (BP)", the top five terms were response to mechanical stimulus, G1/S transition of mitotic cell cycle, cell cycle G1/S phase transition, muscle cell proliferation, mesenchymal cell differentiation; for the "cellular component (CC)", the top five terms were transcription factor complex, cyclindependent protein kinase holoenzyme complex, nuclear chromatin, nuclear transcription factor complex, Flemming body; The top five "molecular function (MF)" terms were transcription factor activity and RNA polymerase II proximal promoter sequence-specific DNA binding, transcriptional activator activity and RNA polymerase II transcription regulatory region sequence-specific DNA binding, proximal promoter sequence-specific DNA binding, transcriptional activator activity and RNA polymerase II proximal promoter sequence-specific DNA binding, transcriptional repressor activity and RNA polymerase II transcription regulatory region sequence-specific DNA binding (Table 2 ). Additionally, KEGG pathway analysis showed that DEGs were enriched in 30 pathways, such as Cell cycle, Small cell lung cancer, p53 signaling pathway, MicroRNAs in cancer, Cellular senescence (Table 2 and Figure 3). Long noncoding RNAs (lncRNAs) are defined as noncoding RNAs longer than 200 nucleotides [16]. lncRNAs were showed to be involved in a variety of biological regulatory functions including metastasis and tumorigenesis of cancer [17,18] Dleu2 could control miR-16-1 to regulate proliferation, invasion and migration of laryngeal cancer [22]. ADAMTS9-AS1 were related to overall survival of breast cancer patients [23], bladder cancer [24] and colon adenocarcinoma [25]. Few studies, however, have explored the relationship between above-mentioned DELs and tumorigenesis of WT.
Therefore, further studies are needed to illuminate the molecular and biological mechanism of these DELs in WT.
MicroRNAs are single-stranded and 18-25 nucleotide-long noncoding RNA which targets mRNAs to control gene expression [26]. DEMs in WT, including 105 up-regulated and 49 down-regulated DEMs, were summarized in present study. In present study, ceRNA network contained 14 differentially expressed miRNAs. However, only one miRNA (miR-200a) were related to overall survival in WT patients. MiR-200a was an important member of miR-200 family. It had been reported that miR-200a was involved in several biological processes to control progression of cancer [27,28]. MiR-200a was showed to target FOXA1 and acts as a tumor suppressor on the survival, proliferation and invasion of glioma cells [28]. And miR-200a might inactivate BRD4-mediated AR signaling to inhibit progression of prostate cancer [27]. Moreover, previous studies reported that miR-200a is associated with the development and occurrence of esophageal cancer, breast cancer and endometrial cancer, breast cancer, and esophageal cancer by targeting specific genes, such as CRMP-1, EPHA2 and PTEN [29][30][31] The major limitation of present study is that tumor tissue and blood verification of these differentially expressed lncRNAs, miRNAs, mRNA and relative pathways is lacking. Further targeted studies related to this ceRNA network still need to be designed to verify and investigate these valuable ncRNAs in the progression of WT.

Conclusion
In summary, differentially expressed lncRNAs, mRNAs, and miRNAs were identified and functional lncRNA-miRNA-mRNA ceRNA regulatory network for WT tumorigenesis was successfully constructed. Significantly altered lncRNAs, mRNAs and miRNAs might serve as prognosis biomarkers and therapeutic targets for tumorigenesis of WT. The ceRNA regulatory network might illuminate inner molecular mechanism which was involved in progression and tumorigenesis of WT.

Ethics approval and consent to participate
The present study, the data download from Therapeutically Applicable Research to Generate Effective Treatment database, therefore, this article does not contain any studies with human participants or animals performed by any of the authors. thus no ethical approval and patient consent are required.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analyzed during the current study download from Therapeutically Applicable Research to Generate Effective Treatment database(https://ocg.cancer.gov/programs/target).

Conflicts of Interests
All authors declare that they have no conflict of interest to state.

Funding
The present study was supported by research start-up fee for the eighth affiliated hospital, Sun Yat-sen University (grant no.zdbykyqdf005).