Identification of the hsa_circ_0039466/miR-96-5p/FOXO1 regulatory network in hepatocellular carcinoma by whole-transcriptome analysis

Feng Yuan; Yongchang Tang; Mingbo Cao; Yupeng Ren; Yuxuan Li; Gaoyuan Yang; Qifeng Ou; Francisco Tustumi; Giovanni Battista Levi Sandri; Driss Raissi; Christine Pocha; Meihai Deng; Zhicheng Yao

doi:10.21037/atm-22-3147

Identification of the hsa_circ_0039466/miR-96-5p/FOXO1 regulatory network in hepatocellular carcinoma by whole-transcriptome analysis

Ann Transl Med. 2022 Jul;10(14):769. doi: 10.21037/atm-22-3147.

Authors

Affiliations

¹ Department of Hepatobiliary Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
² Laboratory of General Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
³ Department of Gastroenterology, Digestive Surgery Division, University of São Paulo Medical School, Sao Paulo, Brazil.
⁴ Department of Surgical Sciences, Sapienza University, Rome, Italy.
⁵ Division of Interventional Radiology, Department of Radiology, University of Kentucky Medical Center, Lexington, KY, USA.
⁶ Avera Hepatology and Transplant Institute, University of South Dakota, Sioux Falls, SD, USA.
⁷ Department of General Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

^# Contributed equally.

Abstract

Background: Circular RNAs (circRNAs) are important for the process of cancer initiation and progression. However, the role of circRNAs in hepatocellular carcinoma (HCC) remains incompletely understood. Therefore, we further explored the expression network of circRNAs in HCC.

Methods: Whole-transcriptome microarrays of HCC and paired normal liver tissues were obtained from the Gene Expression Omnibus (GEO) database. The structures of tumor-associated circRNAs were acquired by the Cancer-Specific CircRNA Database (CSCD). StarBase, circBank, and R packages (miRNAtap and multiMiR) were used to predict miRNA targets of circRNAs and downstream molecules of miRNAs. Expression relationships between RNA-RNA interactions were evaluated by data from The Cancer Genome Atlas (TCGA) and GEO databases. ClusterProfiler and DOSE R packages were used for pathway enrichment to explore the biological functions of potential target genes. Finally, a possible circRNA-miRNA-mRNA regulatory network was established based on the competing endogenous RNA (ceRNA) hypothesis.

Results: The differentially expressed circRNAs (DECs) were matched with cancer-specific circRNAs in the CSCD database and a screening analysis was performed to obtain 5 cancer-specific circRNAs. A total of 329 possible target miRNAs for 5 cancer-specific circRNAs were predicted by the circBank database, and intersection analysis with differentially expressed miRNAs (DEmiRNAs) revealed that miR-6746-3p and miR-96-5p were the two most suitable miRNAs targets for our selected circRNAs. Further expression verification and prediction of base complementary paired binding sites demonstrated the hsa_circ_0039466/miR-96-5p axis as a crucial pathway in HCC. Next, we found that FOXO1 and LEPR were two potential downstream molecules of the hsa_circ_0039466/miR-96-5p axis through target gene prediction analysis, differential expression analysis, and intersection analysis. The pathway enrichment results suggested that the hsa_circ_0039466/miR-96-5p axis affects the progression and outcome of HCC through the insulin resistance pathway. Finally, through multi-data crossover analysis and data analysis of HCC samples further confirmed the existence of the hsa_circ_0039466/miR-96-5p/FOXO1 ceRNA regulatory network and that the axis was closely related to clinical stage.

Conclusions: hsa_circ_0039466 facilitates the expression of FOXO1 by sponging miR-96-5p, and ultimately inhibits tumor progression. These results provide a theoretical basis for further understanding of the gene expression network of HCC.

Keywords: Gene Expression Omnibus (GEO); Hepatocellular carcinoma (HCC); The Cancer Genome Atlas (TCGA); circular RNA hsa_circ_0039466; competitive endogenous RNA (ceRNA).