The consequences of manipulating relaxin family peptide receptor 1 (RXFP1) level in ovarian cancer cells

Abstract

Deregulation of the relaxin family peptide system (RFPS) appears to increase the risk of range of cancers, including epithelial ovarian cancers (EOC). The present study examines the effect of relaxin family peptide receptor 1 (RXFP1) level on the biological properties of human epithelial ovarian adenocarcinoma cells (OVCAR4 and SKOV3). RXFP1 was downregulated (RXFP1↓) in the cells using the RXFP1 sgRNA CRISPR All-in-One Lentivirus set (pLenti-U6-sgRNA-SFFV-Cas9–2A-Puro), and upregulated (RXFP1↑) using the RXFP1 CRISPRa sgRNA Lentivector (pLenti-U6-sgRNA-PGK-Neo) kit, which activates the RXFP1 gene when paired with dCas9-SAM. The changes taking place during adhesion to extracellular matrix (ECM) proteins were assessed in multi-well plates coated with collagen, fibronectin, laminin and gelatin. Cellular viability was monitored based on mitochondrial metabolic activity (MTT Assay, Alamar Blue Assay) and adenosine triphosphate production (ATP Assay). The rate of cell proliferation was determined based on the percentage of Ki67 immunoreactive cells and the numbers of cells in particular cell-cycle phases. The mesenchymal-like (Boyden Chamber Assay) and amoeboid-like movements (Wound Healing Assay) of ovarian cancer cells were also analyzed after transfection. RXFP1 downregulation decreased the adhesion properties of ovarian cancer cells and increased the tendency for apoptosis under stressful conditions. In contrast, RXFP1 upregulation had pro-proliferative, pro-survival and promigratory effects. Our findings confirm that the relaxin-2/RXFP1 signaling pathway plays a role in the promotion of growth and progression of ovarian cancer.