Effect of miR-181c-5p on vasculogenic mimicry in ovarian cancer stem-like cells and its mechanism

Abstract

Objective To investigate the effect of miR-181c-5p on vasculogenic mimicry (VM) in ovarian cancer stem-like cells (OCS-LCs) and its mechanism. Methods OVCAR3 cells were induced into OCS-LCs by serum-free suspension culture. OVCAR3 cells were divided into groups A, B, and C and were transfected with NC-miR-181c-5p, siRNA-miR-181c-5p, and pRNA-miR-181c-5p, respectively. The sphere-forming experiment was used to assess the sphere formation ability of groups A, B, and C. RT-qPCR was used to measure the relative expression level of miR-181c-5p in cells of groups A, B, and C, and Western blot was used to measure the relative protein expression levels of Oct-4, Nanog, HIF-1α, and VEGF in cells of groups A, B, and C. CCK-8 assay was used to measure the viability of cells in groups A, B, and C, and the three-dimensional culture experiment was used to measure the angiogenesis rate of groups A, B, and C. Results OVCAR3 cells were successfully induced into OCS-LCs. RT-qPCR showed that group B had a significantly lower relative expression level of miR-181c-5p than group A, and group C had a significantly higher relative expression level than group A (t=2.25,8.68,P<0.05). The sphere-forming experiment showed that compared with group A, group B had significant decreases in sphere-forming cycle, and increases in maximum sphere diameter, and sphere-forming rate, and compared group A with group C, these indicators had the same changes (t=5.56-33.66,P<0.05). Western blot showed that compared with group A, group B had significant increases in the relative protein expression levels of Oct-4, Nanog, HIF-1α, and VEGF, and compared with group C, group A had significant increases in the relative expression levels of these proteins (t=4.51-56.15,P<0.05). CCK-8 assay showed that group B had a significantly higher cell viability than group A, and group C had a significantly lower cell viability than group A (F=97.70-281.80,P<0.05). The three-dimensional culture experiment showed that group B had a significant increase in angiogenesis rate compared with group A, and compared with group C, group A had a significant increase in angiogenesis rate (t=3.70,18.67,P<0.05). Conclusion This study shows that miR-181c-5p may inhibit VM formation in OCS-LCs by reducing the protein expression levels of HIF-1α and VEGF in cells.

Keywords

• ovarian neoplasms|neoplastic stem cells|micrornas|hypoxia-inducible factor 1, alpha subunit|vascular endothelial growth factors|neovascularization, pathologic|in vitro techniques