CircSPECC1 in cancer-associated fibroblasts promotes invasion and migration of lung adenocarcinoma cells and enhances CCL2 mRNA stability

Objectives Circular RNA (circRNA) is linked to the pathogenesis of various malignancies mediated by cancer-associated fibroblasts (CAFs). However, their role and molecular mechanisms in lung adenocarcinoma (LUAD) remain largely unexplored. Methods CAFs and normal fibroblasts (NFs) isolated from LUAD patients were subjected to RNA sequencing to identify differentially expressed circRNAs. Candidate circRNAs with differential expression were quantitatively validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in CAFs and plasma from LUAD patients. Their prognostic value was assessed by survival analysis using the Kaplan-Meier method. To validate the function of SPECC1 circular RNA (circSPECC1) in CAFs, knockdown and overexpression vectors were constructed. In vitro co-culture systems and functional assays were employed to evaluate the impact of circSPECC1 on the growth and metastatic potential of LUAD cells. Furthermore, RNA pull-down, RNA immunoprecipitation, mass spectrometry, and qRT-PCR were used to investigate the underlying mechanism of circSPECC1. Results Several differentially expressed circRNAs were identified between CAFs and NFs, from which six core circRNAs were selected based on fold-change criteria. The expression patterns of these core circRNAs, as determined by RNA sequencing, were confirmed by qRT-PCR in CAFs (vs. NFs) and in plasma from LUAD patients (vs. normal). Among them, circSPECC1, which we are interested in, showed significant differential expression and was significantly correlated with advanced pathological stages and LUAD cohorts with poor clinical outcomes. Additionally, circSPECC1 is a cytoplasm-localized circRNA generated by back-splicing of exon 4 of the SPECC1 gene. Overexpression of circSPECC1 in CAFs promoted LUAD cell migration and invasion without affecting LUAD cell proliferation. Mechanistically, circSPECC1 was found to bind to heterogeneous nuclear ribonucleoprotein K (hnRNPK) and to enhance the stability of chemokine (C-C motif) ligand 2 (CCL2) mRNA. Conclusion CircSPECC1 is an emerging prognostic indicator in LUAD and is associated with the migration and invasion of LUAD cells induced by CAFs, potentially via its interaction with hnRNPK and modulation of CCL2 mRNA stability. Our study provides novel insights into developing CAF-targeted therapies in LUAD.