Abstract
Emerging discoveries of dynamic and reversible N6-methyladenosine (m6A) modification on RNA in mammals have revealed the key roles of the modification in human tumorigenesis. As known m6A readers, insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are upregulated in most cancers and mediates the enhancement of m6A-modified mRNAs stability. However, the mechanisms of IGF2BPs in renal cell cancer (RCC) still remain unclear. Bioinformatic analysis and RT-qPCR were performed to evaluate the expression of IGF2BPs and m6A writer Wilms tumor 1-associating protein (WTAP) in RCC samples and its correlation with patient prognosis. In vitro, in vivo biological assays were performed to investigate the functions of IGF2BPs and WTAP in RCC. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) combined with bioinformatics analysis and following western blot assay, dual-luciferase reporter assays were performed to validate the regulatory relationships between transcription factor (TF) early growth response 2 (EGR2) and potential target genes IGF2BPs. RNA sequencing (RNA-seq), methylated RNA immunoprecipitation-qPCR (MERIP-qPCR), RIP-qPCR, m6A dot blot, and dual-luciferase reporter assays combined with bioinformatics analysis were employed to screen and validate the direct targets of IGF2BPs and WTAP. Here, we showed that early growth response 2 (EGR2) transcription factor could increase IGF2BPs expression in RCC. IGF2BPs in turn regulated sphingosine-1-phosphate receptor 3 (S1PR3) expression in an m6A-dependent manner by enhancing the stability of S1PR3 mRNA. They also promoted kidney tumorigenesis via PI3K/AKT pathway. Furthermore, IGF2BPs and WTAP upregulation predicted poor overall survival in RCC. Our studies showed that the EGR2/IGF2BPs regulatory axis and m6A-dependent regulation of S1PR3-driven RCC tumorigenesis, which enrich the m6A-modulated regulatory network in renal cell cancer. Together, our findings provide new evidence for the role of N6-methyladenosine modification in RCC.
Highlights
In 2018, 400,000 new diagnoses and over 170,000 deaths due to renal cancer were reported [1]
High Wilms tumor 1-associating protein (WTAP) and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) expression correlate with overall survival of patients with Renal cell carcinoma (RCC) To determine the role of m6A RNA modification in RCC, a heatmap showing expression patterns of m6A regulators across various kidney tissues in the TCGA database was designed (Fig. 1A)
High mRNA levels of IGF2BP2 and IGF2BP3 were associated with late clinical stage, nodal involvement, and distant metastasis
Summary
In 2018, 400,000 new diagnoses and over 170,000 deaths due to renal cancer were reported [1]. Comprises about 90% of all renal-originated tumors [2], with 35% of RCC patients develop metastases [3]. The Wilms tumor 1-associating protein (WTAP) stabilizes MTC localization and substrate recruitment [10,11,12]. Recent studies show that IGF2BPs, an additional family of readers, stabilize the translation of target mRNA, in contrast to YTHDF2 [13, 14]. Binding proteins, IGF2BPs promote tumor initiation and metastasis by stabilizing the m6A-containing mRNAs [17,18,19,20,21]. Few studies have explored the role of IGF2BPs in renal cancer
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