circ-RHOT1 Knockdown Suppresses Malignant Phenotypes and Reduces Stemness-Associated Marker Expression in Triple-Negative Breast Cancer Cells through PRMT5

To determine whether circ-RHOT1 regulates malignant phenotypes, oxidative stress, and stemness-associated marker expression in triple-negative breast cancer (TNBC) through PRMT5. The expression levels of circ-RHOT1 and PRMT5 were examined in 32 paired TNBC and adjacent normal tissues, the normal mammary epithelial cell line MCF-10 A, and the TNBC cell lines MDA-MB-231 and MDA-MB-468. Circ-RHOT1 was silenced in MDA-MB-231 and MDA-MB-468 cells, followed by PRMT5 overexpression-mediated rescue experiments. RT-qPCR and western blotting were used to determine RNA and protein expression, respectively. Cell viability, clonogenic growth, apoptosis, migration, and invasion were assessed using CCK-8, colony formation, flow cytometry, and Transwell assays. Intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels were measured to evaluate oxidative stress. The expression of the stemness-associated markers CD133, CD44, and BMI1 was assessed by RT-qPCR. Circ-RHOT1 and PRMT5 were upregulated in TNBC tissues and cell lines, and their expression levels were positively correlated in TNBC tissues. Circ-RHOT1 knockdown reduced PRMT5 protein abundance and consistently suppressed cell viability, clonogenic growth, migration, and invasion in MDA-MB-231 and MDA-MB-468 cells, while increasing apoptosis. Circ-RHOT1 knockdown also increased ROS and MDA levels, decreased SOD activity, and reduced CD133, CD44, and BMI1 expression. PRMT5 overexpression partially reversed these changes in both TNBC cell lines. These findings support PRMT5 as a downstream mediator of circ-RHOT1-associated malignant phenotypes in TNBC cells and link the circ-RHOT1/PRMT5 axis to oxidative stress and stemness-associated marker expression. Further mechanistic, functional, and in vivo validation is required to establish its therapeutic relevance.