The Regulatory Network and Role of the circRNA-miRNA-mRNA ceRNA Network in the Progression and the Immune Response of Wilms Tumor Based on RNA-Seq

Abstract

Circular RNA (circRNA), which is a newly discovered non-coding RNA, has been documented to play important roles in miRNA sponges, and the dysregulation of which is involved in cancer development. However, circRNA expression profiles and their role in initiation and progression of Wilms tumor (WT) remain largely unclear at present. Here, we used paired WT samples and high-throughput RNA sequencing to identify differentially expressed circRNAs (DE-circRs) and mRNAs (DE-mRs). A total of 314 DE-circRs and 1612 DE-mRs were identified. The expression of a subset of differentially expressed genes was validated by qRT–PCR. A complete circRNA-miRNA-mRNA network was then constructed based on the common miRNA targets of DE-circRs and DE-mRs identified by miRanda prediction tool. The Gene set enrichment analysis (GSEA) indicated that several signaling pathways involving targeted DE-mRs within the ceRNA network were associated with cell cycle and immune response, which implies their participation in WT development to some extent. Subsequently, these targeted DE-mRs were subjected to implement PPI analysis and to identify 10 hub genes. Four hub genes were closely related to the survival of WT patients. We then filtered prognosis-related hub genes by Cox regression and least absolute shrinkage and selection operator (LASSO) regression analysis to construct a prognosis-related risk score system based on a three-gene signature, which showed good discrimination and predictive ability for WT patient survival. Additionally, we analyzed the mutational landscape of these genes and the associations between their expression levels and those of immune checkpoint molecules and further demonstrated their potential impact on the efficacy of immunotherapy. qRT–PCR and western blotting (WB) analysis were used to validate key differentially expressed molecules at the RNA and protein levels, respectively. Besides these, we selected a key circRNA, circEYA1, for function validation. Overall, the current study presents the full-scale expression profiles of circRNAs and the circRNA-related ceRNA network in WT for the first time, deepening our understanding of the roles and downstream regulatory mechanisms of circRNAs in WT development and progression. We further constructed a useful immune-related prognostic signature, which could improve clinical outcome prediction and guide individualized treatment.

CCNB1 is a novel prognostic biomarker and promotes proliferation, migration and invasion in Wilms tumor

“…The Illumina NovaSeq 6000 was used for library construction and sequencing at Shanghai Sinomics Corporation (Illumina, USA). Detailed procedures can be referred to the previously published paper [ 18 ].…”

“…Cellular RNA was extracted from WT cells (WIT-49) and normal renal epithelial cells (293T). RT-PCR methodology was performed according to previous literature [ 18 ]. The expression of CCNB1 and GAPDH was based on the formula 2 ^-ΔΔCt .…”

Immune-related gene signature associates with immune landscape and predicts prognosis accurately in patients with Wilms tumour

“…Total cellular RNA from three WT cells (Wit-49, WT-CLS1, and SK-NEP-1) and normal renal epithelial cells (293T) were obtained using the Simply P Total RNA Extraction Kit (BioFlux, China) following the manufacturer’s guidelines. The methodology of RT-PCR was performed in accordance with the previous literature ( 37 ). The expression of candidate genes (NRP2, EGF, NODAL, and NR2F2) was based on the formula 2^ -ΔΔCt .…”

Functions of Circular RNA in Human Diseases and Illnesses

“…As previously mentioned, an innate immune response occurs when cellular circRNAs are degraded by endonuclease RNase L release PKR [ 120 ]. Several targeted DE-mRs signaling pathways found in the ceRNA network are involved in the evolvement of Wilms tumors (WT) because these pathways are linked with immune response and cell cycle [ 123 ].…”