Mapping a Circular RNA–microRNA–mRNA-Signaling Regulatory Axis that Modulates Stemness Properties of Cancer Stem Cell Populations in Colorectal Cancer Spheroid Cells

Abstract

Spheroidal cancer cell cultures have been used to enrich cancer stem cells (CSC), which are thought to contribute to important clinical features of tumors. This study aimed to map the regulatory networks driven by circular RNAs (circRNAs) in CSC-enriched colorectal cancer (CRC) spheroid cells. The spheroid cells established from two CRC cell lines acquired stemness properties in pluripotency gene expression and multi-lineage differentiation capacity. Genome-wide sequencing identified 1503 and 636 circRNAs specific to the CRC parental and spheroid cells, respectively. In the CRC spheroids, algorithmic analyses unveiled a core network of mRNAs involved in modulating stemness-associated signaling pathways, driven by a circRNA–microRNA (miRNA)–mRNA axis. The two major circRNAs, hsa_circ_0066631 and hsa_circ_0082096, in this network were significantly up-regulated in expression levels in the spheroid cells. The two circRNAs were predicted to target and were experimentally shown to down-regulate miR-140-3p, miR-224, miR-382, miR-548c-3p and miR-579, confirming circRNA sponging of the targeted miRNAs. Furthermore, the affected miRNAs were demonstrated to inhibit degradation of six mRNA targets, viz. ACVR1C/ALK7, FZD3, IL6ST/GP130, SKIL/SNON, SMAD2 and WNT5, in the CRC spheroid cells. These mRNAs encode proteins that are reported to variously regulate the GP130/Stat, Activin/Nodal, TGF-β/SMAD or Wnt/β-catenin signaling pathways in controlling various aspects of CSC stemness. Using the CRC spheroid cell model, the novel circRNA–miRNA–mRNA axis mapped in this work forms the foundation for the elucidation of the molecular mechanisms of the complex cellular and biochemical processes that determine CSC stemness properties of cancer cells, and possibly for designing therapeutic strategies for CRC treatment by targeting CSC.

Regulation of metabolic homeostasis by the TGF‐β superfamily receptor ALK7

“…Due to the similarities between ALK7, ALK4, and ALK5 discussed above, over-expression of ALK7 will result in activation of pathways that are common to all these receptors, hence not possible to exclusively ascribe to endogenously expressed ALK7. Several microRNAs with effects on proliferation, apoptosis, or invasion of cancer cells have also been reported to target the mRNA encoding ALK7, including miR-382 and miR-548c-3p [53], miR-23b [43], miR-454 [54], and miR-376c [55]. However, these microRNAs have multiple target genes, including other TGF-b superfamily receptors [56], and hence, the specific contribution of ALK7 to the effects reported following microRNA overexpression or knock-down remains unclear.…”

Non-Coding RNA and Frizzled Receptors in Cancer

“…In chronic lymphocytic leukemia (CLL) cells, circ-CBFB indirectly upregulates Fzd3 expression and downstream Wnt/β-catenin signaling by inhibiting miR-607 ( Xia et al, 2018 ). Similar mechanisms were observed in CRC spheroid cells where circ_0082096 and circ_006631 upregulate Fzd3 expression by inhibiting miR382, miR-579, miR-224, and miR-548c ( Rengganaten et al, 2020 ). CircRNA_100290 also influences colorectal carcinoma (CRC) by inhibiting miR-516b in CRC cells, indirectly upregulating Fzd4 and the Wnt/β-catenin pathway ( Fang et al, 2018 ).…”

Pepsinogen C expression–related lncRNA/circRNA/mRNA profile and its co-mediated ceRNA network in gastric cancer

“…Of these, the top three with the highest degree score of DEcircRNAs included hsa_circ_0031583, hsa_circ_0008197, and hsa_circ_0036627. A ceRNA network mediated via DEcircRNAs modulated histological classi cations and gastric cancer progression (29) and the stemness properties of colorectal cancer stem cells (30). No report has shown that hsa_circ_0031583, hsa_circ_0008197, and hsa_circ_0036627 affected tumor progression.…”