Mechanism study on circASAP1 promoting proliferationand inhibiting apoptosis of osteosarcoma cells by targeting miR-4500

Objective To investigate the regulatory effect of circular RNA ASAP1 (circASAP1) targeting microRNA-4500 (miR-4500) on the proliferation and apoptosis of osteosarcoma cells.

Methods Human osteosarcoma cells (Saos-2) were selected as the experimental subjects and cultured in DMEM medium containing 10% fetal bovine serum while maintaining 5% CO₂. The expressions of circASAP1 and miR-4500 in osteosarcoma tissues were detected using RT-qPCR. Saos-2 cells were transfected with si-NC (40 nmol/L), si-circASAP1 (40 nmol/L), pcDNA (0.4 μg), pcDNA-circASAP1 (0.4 μg), miR-NC (40 nmol/L), miR-4500 mimics (40 nmol/L), si-circASAP1+anti-miR-NC (40 nmol/L), and si-circASAP1+anti-miR-4500 (40 nmol/L), respectively. Cell proliferation and apoptosis were assessed using CCK-8, colony formation assays, and flow cytometry. The interaction between circASAP1 and miR-4500 was analyzed using a dual-luciferase reporter assay.

Results Compared with adjacent non-tumor tissues, the expression of circASAP1 was significantly upregulated, while that of miR-4500 was significantly downregulated in osteosarcoma tissues (P < 0.001). Compared with the si-NC group, the si-circASAP1 group exhibited significantly decreased circASAP1 expression, optical density (OD) values, and the number of cell colonies (P < 0.001). Compared with the si-NC group, the si-circASAP1 group showed significantly upregulated levels of cleaved-caspase3 protein, cleaved-caspase9 protein, and the apoptosis rate (P < 0.001). StarBase search revealed specific binding sequences between miR-4500 and circASAP1. Co-transfection of miR-4500 mimics and WT-circASAP1 significantly reduced the relative luciferase activity of the cells(0.34±0.03) versus (0.95±0.06), t=27.280, P < 0.001. The expression of miR-4500 in the pcDNA-circASAP1 group was significantly lower than that in the pcDNA group, whereas the expression of miR-4500 in the si-circASAP1 group was significantly higher than that in the si-NC group (P < 0.001). Compared with the miR-NC group, the miR-4500 group exhibited significantly increased miR-4500 expression, cleaved-caspase3 protein levels, cleaved-caspase9 protein levels, and the apoptosis rate, while the OD values and the number of colonies significantly decreased (P < 0.001). Compared with the si-circASAP1+anti-miR-NC group, the si-circASAP1+anti-miR-4500 group showed significantly decreased miR-4500 expression, cleaved-caspase3 protein levels, cleaved-caspase9 protein levels, and the apoptosis rate, while the OD values and the number of colonies significantly increased (P < 0.001).

Conclusion The expression of circASAP1 is increased, while that of miR-4500 is decreased in osteosarcoma tissues. Moreover, circASAP1 promotes the proliferation and inhibits the apoptosis of osteosarcoma cells by targeting miR-4500.