Hsa_circ_0000231 activates the MAPK pathway via IGF2BP2-mediated CACNA1H mRNA stabilization to promote cervical...

Circular RNAs (circRNAs) are key regulators in tumorigenesis, including cervical cancer (CC), but their specific roles and molecular mechanisms in CC remain incompletely understood. This study further explored the functional role of hsa_circ_0000231 in CC cells and its underlying molecular mechanisms.

The circular nature of circ_0000231 was confirmed using Sanger sequencing, divergent primer PCR, RNase R digestion assay, and subcellular fractionation assay. Gain-of-function experiments involved transfecting SiHa and HeLa cells with circ_0000231-overexpressing plasmids, whereas loss-of-function experiments utilized shRNA-mediated silencing of circ_0000231 in MS751 cells and siRNA-mediated knockdown of CACNA1H in SiHa and HeLa cells. Gene expression levels were verified by qPCR. RNA immunoprecipitation (RIP) assay was used to confirm the direct interactions of circ_0000231 and CACNA1H with IGF2BP2, and mRNA stability assay was employed to evaluate the effect of circ_0000231 and IGF2BP2 on CACNA1H mRNA stability. Cell proliferation, cycle distribution, apoptosis, migration, and invasion were analyzed using MTT, flow cytometry, and Transwell assays, respectively. mRNA transcriptome sequencing was performed to identify differentially expressed genes (DEGs) in CC cells after circ_0000231 overexpression, and protein levels were detected by Western blot.

Compared with non-tumorigenic immortalized human keratinocyte Hacat cells, hsa_circ_0000231 was upregulated in most CC cell lines (HeLa, SiHa, MS751) but not in Caski cells. It exhibited resistance to RNase R digestion and was predominantly localized in the cytoplasm of MS751 cells. Overexpression of circ_0000231 promoted cell proliferation, cell cycle progression, migration, and invasion, while inhibiting apoptosis in CC cells. Conversely, silencing circ_0000231 in MS751 cells exerted the opposite effects. mRNA transcriptome sequencing and KEGG pathway analysis identified the MAPK pathway as an enriched pathway. Intersection analysis of DEGs in HeLa and SiHa cells revealed that CACNA1H, a MAPK pathway-related gene, was upregulated in circ_0000231-overexpressing cells. Silencing CACNA1H reversed the pro-tumor effects of circ_0000231 overexpression, specifically attenuating MAPK pathway activation, restoring apoptosis, and inhibiting cell viability, migration, and invasion. Furthermore, RIP assay confirmed that both circ_0000231 and CACNA1H could bind to IGF2BP2. Knockdown of circ_0000231 or IGF2BP2 significantly reduced the mRNA stability of CACNA1H in MS751 cells, indicating that the hsa_circ_0000231/IGF2BP2 axis enhances CACNA1H mRNA stability.

Hsa_circ_0000231 acts as an oncogenic circRNA in CC by promoting cell proliferation, migration, and invasion, and activating the MAPK pathway via CACNA1H. Mechanistically, circ_0000231 regulates CACNA1H mRNA stability through binding to IGF2BP2, which clarifies the molecular mechanism underlying circ_0000231-mediated CC progression. These findings extend the current understanding of circ_0000231 regulatory networks in CC and highlight circ_0000231 as a potential therapeutic target.