Integrated whole transcriptome profiling revealed a convoluted circular RNA-based competing endogenous RNAs regulatory network in colorectal cancer

www.nature.com/scientificreports OPEN Integrated whole transcriptome profiling revealed a convoluted circular RNA‑based competing endogenous RNAs regulatory network in colorectal cancer Hasan Mollanoori 1, Yaser Ghelmani 2,4, Bita Hassani 3,4 & Mohammadreza Dehghani 1* Recently, it has been identified that circRNAs can act as miRNA sponge to regulate gene expression in various types of cancers, associating them with cancer initiation and progression. The present study aims to identify colorectal cancer-related circRNAs and the underpinning mechanisms of circRNA/miRNA/mRNA networks in the development and progress of Colorectal Cancer. Differentially expressed circRNAs, miRNAs, and mRNAs were identified in GEO microarray datasets using the Limma package of R. The analysis of differentially expressed circRNAs resulted in 23 upregulated and 31 downregulated circRNAs. CeRNAs networks were constructed by intersecting the results of predicted and experimentally validated databases, circbank and miRWalk, and by performing DEMs and DEGs analysis using Cytoscape. Next, functional enrichment analysis was performed for DEGs included in ceRNA networks. Followed by survival analysis, expression profile assessment using TCGA and GEO data, and ROC curve analysis we identified a ceRNA sub-networks that revealed the potential regulatory effect of hsa_circ_0001955 and hsa_circ_0071681 on survival-related genes, namely KLF4, MYC, CCNA2, RACGAP1, and CD44. Overall, we constructed a convoluted regulatory network and outlined its likely mechanisms of action in CRC, which may contribute to the development of more effective approaches for early diagnosis, prognosis, and treatment of CRC. Colorectal cancer (CRC) accounts for 10 percent of all new cancers cases and 9.4 percent of cancer-related mortality around the world, ranking it third in terms of incidence and second in terms of mortality1. Despite significant advancements in understanding CRC, it was estimated that over 1.9 million new CRC cases and 935,000 deaths occurred in 2020, projected to rise to more than 2.2 million new cases and 1.1 million deaths by 20301,2. Moreover, the mortality rate remains high, with an overall 5-year survival rate of only 50%, primarily due to late detection, high recurrence rates, and distant m etastasis3–5. Detecting CRC at an early and localized stage significantly increases the 5-year survival rate to 90% and facilitates the identification and removal of adenomas, which can lead to colorectal cancer. So, developing methods for early detection is crucial in preventing colorectal c ancer6. Circular RNAs (circRNAs) belong to a newly discovered class of non-coding RNAs lack the 5′-end cap and 3′-end ploy A tail. They form covalently closed-continuous loop by linking the 5′ and 3′ ends generated through back splicing pre-mRNAs. This closed-loop structure confers circRNAs with resistance to exonucleases, making them more stable than linear t ranscripts7,8. Recent studies have demonstrated the different roles of circRNAs in cancer, including their involvement in cell proliferation, tumor metastasis, and drug resistance9. Among these roles, the most indicated function for circRNAs is acting as MicroRNA (miRNA) sponges. The competing endogenous RNA (ceRNA) hypothesis, first presented by Salmena et al., suggests that circRNAs efficiently sequester miRNAs, preventing their function and positively regulating the expression of their mRNA targets due to the shared miRNA response elements (MRE)10. Several studies have reported the dysregulation of circRNAs 1 Medical Genetics Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2Clinical Research Development Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 3Sarem Gynecology, Obstertrics and Infertility Research Center, Sarem Women’s Hospital, Iran University of Medical Sciences (IUMS), Tehran, Iran. 4These authors contributed equally: Yaser Ghelmani and Bita Hassani. *email: Scientific Reports | (2024) 14:91 | https://doi.org/10.1038/s41598-023-50230-0 1 Vol.:(0123456789) www.nature.com/scientificreports/ as a possible mechanism contributing to the development of CRC11–14. For instance, in CRC, overexpression of ciRS-7-a suppresses miR-7, thus alleviating the miR-7-mediated suppression of the EGFR/RAF1/MAPK pathway, a well-known oncogenic pathway that correlates with poor survival and m etastasis12. Apart from their close association with cancer development, circRNAs’ stability, conservation, and abundance in body fluids also make them valuable diagnostic biomarkers and potential therapeutic targets for cancer and other human d iseases15. In the present study, we utilized an integrated Systems Biology approach to construct circRNA-miRNAmRNA ceRNA regulatory networks for CRC based on ceRNA hypothesis. We employed publicly available Gene Expression Omnibus (GEO) datasets for this purpose, and subsequently, we further assessed and refined our findings using data from the Cancer Genome Atlas (TCGA). To the best of our knowledge, this study represents the most comprehensive investigation providing potential circRNA-based ceRNA networks specific to CRC. The suggested ceRNAs regulatory networks derived from our study offer a novel perspective into the underlying molecular mechanisms driving CRC development, and they hold promise as innovative diagnostic/prognostic biomarkers and therapeutic targets. Results Identification of differentially expressed circRNAs, miRNAs, and genes We consider circRNAs as a central element in constructing the competing endogenous RNA (ceRNA) network. The publicly available dataset GSE126094 provides expression profiles of 4012 circRNAs in 10 colorectal cancer (CRC) tissues and their corresponding normal-appearing tissues (NATs). Following hierarchical clustering, background correction, and normalization using limma, we conducted differential expression analysis with a cutoff value of an adjusted p value < 0.05 and a |log2 fold change (L2FC)|> 2.0 by limma. This analysis identified 54 significantly differentially expressed circRNAs (DECs), comprising 23 upregulated DECs and 31 downregulated DECs (Fig. 1A). These DECs exhibited distinct expression patterns in tumor tissues compared to normal tissues. Similarly, for miRNA analysis in GSE115513, after hierarchical clustering, background correction, and normalization, we conducted differential expression analysis with a cutoff value of an adjusted p value < 0.05 and a |log2 fold change (L2FC)|> 0.5 using limma. This revealed 19 differentially expressed miRNAs (DEMs), with 14 upregulated DEMs and five downregulated DEMs (Fig. 1B). These miRNAs also showed distinct expression patterns in tumor tissues compared to normal tissues. Furthermore, using GSE39582 as a discovery dataset, after hierarchical clustering, background correction, and normalization, we identified 2084 differentially expressed genes (DEGs) in tumor tissu (...truncated)