Identification of CDC20 as a Novel Biomarker in Diagnosis and Treatment of Wilms Tumor.

Qinlin Shi,Guanghui Wei,Yonglin Li,Tao Lin,Yanping Li,Dawei He,Bo Tang

doi:10.3389/fped.2021.663054

Abstract

Objective: Wilms tumor (WT) is a common malignant solid tumor in children. Many tumor biomarkers have been reported; however, there are poorly targetable molecular mechanisms which have been defined in WT. This study aimed to identify the oncogene in WT and explore the potential mechanisms.Methods: Differentially expressed genes (DEGs) in three independent RNA-seq datasets were downloaded from The Cancer Genome Atlas data portal and the Gene Expression Omnibus database (GSE66405 and GSE73209). The common DEGs were then subjected to Gene Ontology enrichment analysis, protein–protein interaction (PPI) network analysis, and gene set enrichment analysis. The protein expression levels of the hub gene were analyzed by immunohistochemical analysis and Western blotting in a 60 WT sample. The univariate Kaplan–Meier analysis for overall survival was performed, and the log-rank test was utilized. A small interfering RNA targeting cell division cycle 20 (CDC20) was transfected into G401 and SK-NEP-1 cell lines. The Cell Counting Kit-8 assay and wound healing assay were used to observe the changes in cell proliferation and migration after transfection. Flow cytometry was used to detect the effect on the cell cycle. Western blot was conducted to study the changes of related functional proteins.Results: We commonly identified 44 upregulation and 272 downregulation differentially expressed genes in three independent RNA-seq datasets. Gene and pathway enrichment analyses of the regulatory networks involving hub genes suggested that cell cycle changes are crucial in WT. The top 15 highly connected genes were found by PPI network analysis. Furthermore, we demonstrated that one candidate biomarker, CDC20, for the diagnosis of WT was detected, and its high expression predicted poor prognosis of WT patients. Moreover, the area under the curve value obtained by receiver operating characteristic curve analysis from paired WT samples was 0.9181. Finally, we found that the suppression of CDC20 inhibited proliferation and migration and resulted in G2/M phase arrest in WT cells. The mechanism may be involved in increasing the protein level of securin, cyclin B1, and cyclin AConclusion: Our results suggest that CDC20 could serve as a candidate diagnostic and prognostic biomarker for WT, and suppression of CDC20 may be a potential approach for the prevention and treatment of WT.

Highlights

Wilms tumor (WT) is a common pediatric solid retroperitoneal tumor
We identified 3,940 differentially expressed mRNAs, including 2,118 upregulated mRNAs and 1,822 upregulated mRNAs from TCGA database (Figure 1F)
As determined by the CCK8 assay, si-CDC20-1 and si-CDC20-3 significantly slowed cell proliferation in a time-dependent manner in G401and SK-NEP1 cells compared with the cells transfected with negative control (NC) siRNA and siCDC20-2 (p < 0.001, Figures 4A,B). These results indicate that si-CDC20 could decrease WT cell proliferation

Summary

Introduction

The incidence of WT was ∼6 per 100,000 to 7 per 100,000 for children younger than 15 years [1, 2]. Thanks to the continuous efforts by the Children’s Oncology Group and the National Wilms Tumor Society (NWTS), the overall survival rate of WT has improved from 30 to 90% in the last 30 years [3]. Chronic health conditions secondary to treatment impact nearly one quarter of survivors of WT and include renal failure, infertility, cardiac toxicity, restrictive pulmonary disease, and the development of subsequent malignancies [5, 6]. Finding a novel strategy for the diagnosis and treatment of WT has become a hotspot in recent years. A recent whole-exome study has identified that DROSHA and DICER1 mutations impair expression of tumor-suppressing miRNAs [10]. The frequency of alterations in genes is uncommon, and there is no clear gene for clinical application [11]

Methods

Results

Conclusion