RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor

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RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor

Biomedical Engineering | 更新时间：2026-01-09

- RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor
- Journal of Army Medical University Vol. 44, Issue 19, Pages: 2002-2016(2022)
- 作者机构：
  
  400014 重庆，重庆医科大学附属儿童医院泌尿外科，儿童泌尿生殖发育与组织工程重点实验室，儿童发育疾病研究教育部重点实验室，国家儿童健康与疾病临床医学研究中心，儿童发育重大疾病国家国际科技合作基地，儿科学重庆市重点实验室
- 作者简介：
- 基金信息：
  
  the Natural Science Foundation of Chongqing(cstc2021jcyj-msxmX0345);the General Project of Clinical Medical Research of Children's Hospital of Chongqing Medical University(NCRC-2019-GP-08);the Joint Medical Scientific Research Project of Chongqing Science and Technology Commission and Chongqing Health Commission(2022GDRC009)
- DOI：10.16016/j.2097-0927.202204222
  CLC： R318.04;R394.3;R737.11
- Received：29 April 2022，
  
  Revised：2022-06-06，
  
  Published：15 October 2022
- 稿件说明：
移动端阅览
Bin XIANG, Xiaomao TIAN, Tao MI, et al. RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor[J]. Journal of Army Medical University, 2022, 44(19): 2002-2016.
DOI：

Bin XIANG, Xiaomao TIAN, Tao MI, et al. RNA sequencing reveals a regulatory network of circRNA-miRNA-mRNA ceRNA in Wilms tumor[J]. Journal of Army Medical University, 2022, 44(19): 2002-2016. DOI： 10.16016/j.2097-0927.202204222.

摘要

目的

2

基于高通量RNA测序技术和生物信息学分析鉴定肾母细胞瘤(wilms tumor，WT)中潜在的circRNA-miRNA-mRNA调控网络。

方法

2

收集2021年6月至2022年3月期间重庆医科大学附属儿童医院泌尿外科的WT患者的肿瘤切除组织共25例。RNA测序技术分析4对配对的肿瘤和癌旁正常组织的circRNA(DEcircRNA)和mRNA(DEmRNA)差异表达谱。对差异分子进行基于miRanda的miRNA靶点预测，基于相同位点和协同表达建立完整的circRNA-miRNA-mRNA调控网络。最后验证关键环状RNA的功能。

结果

2

筛选出314个DEcircRNAs和1 612个DEmRNAs。通过RT-qPCR在组织中随机选择12个circRNA进行表达量验证，其中7个circRNA显示出与测序结果一致的趋势。基因本体论(gene ontology，GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes，KEGG)分析提示DEcircRNA的亲本基因和DEmRNA主要富集到细胞增殖、癌症和多种代谢相关通路中。利用miRanda预测工具识别DEcircRNA和DEmRNA的共同miRNA靶点，以此构建完整的circRNA-miRNA-mRNA网络。基因集富集分析(Gene Set Enrichment Analysis，GSEA)提示ceRNA网络中的靶基因与细胞周期和免疫应答有关。为了鉴定关键的下游靶点，基于STRING数据库建立了127个ceRNA网络中靶基因的PPI(protein-protein interaction)网络，并进一步确定了前10个HUB基因。其中，4个HUB基因(

TP

53、

KANK

3、

LLGL

1和

TOPA

3)被证实与WT的预后密切相关。基于具有预后意义的关键靶基因构建circRNA作用的调控子网络。最后，通过CCK-8、划痕、Transwell和流式细胞实验，发现沉默子网络中的circRNA(hsa_circ_0009035)抑制了WT细胞的增殖、迁移、侵袭和细胞周期分布(

P

<

0.05)。

结论

2

本研究展示了WT中circRNAs的全面表达，构建了潜在的circRNA相关的ceRNA调控网络，深入阐释了WT发生发展的调控机制。

Abstract

Objective

2

To identify the potential circRNA-miRNA-mRNA regulatory networks in Wilms tumor (WT) based on high-throughput RNA sequencing technology and bioinformatics analysis.

Methods

2

A total of 25 WT specimens were collected from the patients undergoing surgical treatment in our department during June 2021 and March 2022. The differential expression profiles of circRNA (DEcircRNA) and mRNA (DEmRNA) of 4 paired tumor and adjacent normal tissues were analyzed by RNA sequencing. Then

these differential molecules were subjected to miRanda-based miRNA target prediction to establish a complete circRNA-miRNA-mRNA regulatory network based on identical loci and co-expression. Finally

the function of the key circRNAs was validated.

Results

2

We screened 314 DEcircRNAs and 1 612 DEmRNAs out. Of these

12 circRNAs were randomly selected by RT-qPCR in the tumor tissues to make expression verification

and 7 of them showed a trend consistent with the sequencing results. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses suggested that the parental genes of DEcircRNAs and DEmRNAs were mainly enriched in cell proliferation

cancer

and various metabolism-related pathways. The miRanda prediction tool identified common miRNA targets of DEcircRNAs and DEmRNAs as a means to construct a complete circRNA-miRNA-mRNA network. Gene set enrichment analysis (GSEA) suggested that the target genes in the ceRNA network were associated with cell cycle and immune response. To identify key downstream targets

a protein-protein interaction (PPI) network of 127 target genes in the ceRNA network was established based on the STRING database

and 10 hub genes were further identified. Among them

4 hub genes (TP53

KANK3

LLGL1 and TOPA3) were confirmed to be closely associated with the prognosis of WT. Subsequently

a regulatory sub-network of circRNA action was constructed based on the key target genes with prognostic significance. Finally

silencing of circRNA (hsa_circ_0009035) in the sub-network was found to significantly inhibit the proliferation

migration

invasion and cell cycle distribution of WT cells by CCK-8 assay

cell scratch assay

Transwell assay and flow cytometry

respectively (

P

<

0.05).

Conclusion

2

The comprehensive expression of circRNAs in WT is revealed and a potential circRNA-associated ceRNA network is constructed

which profoundly clarify the regulatory mechanism of the occurrence and development of WT.

关键词

Keywords

references

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