Circ-CCDC66 promoted breast cancer progression via miR-4259-mediated upregulation of CCDC66

Circ-CCDC66 (circular-Coiled-coil domain-containing protein 66) has been implicated in therapy resistance in colorectal and gastric cancers, but its role in breast cancer remains poorly understood.

This study aimed to explore the functional role and underlying mechanism of circ-CCDC66 in breast cancer proliferation and stem cell spheroidization.

The circular structure of circ-CCDC66 was verified using agarose gel electrophoresis and Sanger sequencing. Gene expression was assessed by RT-qPCR. Functional assays, including CCK-8 (Cell Counting Kit-8), colony formation, flow cytometry, and spheroidization, were performed following circ-CCDC66 overexpression in breast cancer cells. RNA sequencing and bioinformatics tools (LNCeVar, starBase, LncACTdb 2.0, Cytoscape) were used to construct a circ-CCDC66-miR-4259-CCDC66 regulatory network. Western blotting, luciferase reporter assays, and co-immunoprecipitation-qPCR (CO-IP-qPCR) were used to confirm molecular interactions.

Circ-CCDC66 was highly expressed in breast cancer tissues and cell lines, particularly in MDA-MB-468 and MDA-MB-231 cells. Overexpression of circ-CCDC66 significantly promoted breast cancer cell proliferation and stem cell spheroidization. Mechanistically, circ-CCDC66 acted as a molecular sponge for miR-4259, thereby upregulating CCDC66 expression. This regulatory axis was confirmed through luciferase and CO-IP-qPCR assays.

circ-CCDC66 promotes breast cancer progression by regulating the miR-4259/CCDC66 axis. This novel pathway may serve as a potential therapeutic target for breast cancer treatment.