Neuropilin-1 Knockout and Rescue Confirms Its Role to Promote Metastasis in MDA-MB-231 Breast Cancer Cells.

Breast cancer (BC) metastasis remains a leading cause of female mortality. Neuropilin-1 (NRP-1) is a glycoprotein receptor that plays ligand-dependent roles in BC. Clinical studies indicate its correlation with metastatic disease; however, its functional role in BC metastasis remains uncertain. CRISPR-Cas9 was used to knockout the gene in MDA-MB-231 BC cells, and the effects on metastasis were determined using an orthotopic mouse engraftment model. expression in knockout cells was rescued using a recombinant cDNA with a silent mutation in the sgRNA target-adjacent PAM sequence. Differentially expressed genes between knockout and control cells were determined using whole-transcriptome sequencing and validated using real-time PCR. cells showed a pronounced reduction in the metastasis to the lungs. KEGG pathway analysis of the transcriptome data revealed that PI3K and ECM receptor interactions were among the top altered pathways in the NRP-1KO cells. In addition, reduction in metastasis enhancers proteins, Integrin-β3 and Tenascin-C, and genes and and upregulation of metastasis suppressor genes, and in KO were detected. These findings provide evidence for a functional role for NRP-1 in BC metastasis, supporting further exploration of NRP-1 and the identified genes as targets in treating metastatic BC.