MiRNA-3163 limits ovarian cancer stem-like cells via targeting SOX-2 transcription factor

Abstract

Cancer stem cells (CSCs) are pivotal in both cancer progression and the acquisition of drug resistance. MicroRNAs (miRNAs) play a crucial role in modulating CSC properties and are being explored as potential targets for therapeutic interventions. MiR-3163 is primarily known for its tumor suppressive properties in various human malignancies, with lower expression reported across different cancer types. However, its role in regulating the ovarian CSC phenotype and the underlying mechanism remain largely unknown. Here, we report a remarkable downregulation of miR-3163 in ovarian cancer stem-like cells (CSLCs). Enforced expression of miR-3163 in ovarian adherent and CSLCs, significantly disrupts the stemness phenotype. Moreover, downregulation of miR-3163 expression in ovarian cancer cells (OV2008 and OVCAR-3) inhibits the stem-like cells characterized by CD44+CD117+ expression. Sphere formation assay results reveal that overexpression of miR-3163 in ovarian cancer cells significantly inhibits spheroid formation ability, confirming the regulatory properties of miR-3163 on ovarian CSLCs. Mechanistic investigation reveals that miR-3163 depletes ovarian CSLCs via targeting SOX-2. Furthermore, we establish SOX-2 as a direct target of miR-3163 through dual-luciferase assay. Taken together, our study demonstrates that overexpression of miR-3163 could be a promising strategy for efficiently eradicating the CSC population to prevent chemoresistance and tumor relapse in ovarian cancer patients.

Keywords: Cancer stem-like cells; MicroRNA; Migratory potential; Ovarian cancer.

Fig. 1

Clinical significance of SOX2 expression in ovarian cancer patients (A.) The relative SOX2 expression level in normal vs tumor tissue (TNMplot). (B.) The relative transcripts per million of SOX2 among different cancer stages in ovarian cancer. (C. D.) The mutation count, and SOX2 mRNA expression level with the copy number variation data were extracted from GISTIC. (E.) Gene effect score for SOX2 in different ovarian cancer cell lines (DepMAp online portal).

Fig. 2

Pan cancer and cell line-based analysis of miR-3163 levels. (A.) Pan cancer kaplan meier survival analysis for the miR-3163 levels from TCGA database (CancerMIRNome database). (B.) Extracellular miR-3163 levels from selected circulating miRNome dataset. (C.) The expression analysis of miR-3163 in bulk and CSCs of OV2008 and OVCAR-3 cell lines using real-time PCR

Fig. 3

MiRNA-3163 negatively regulates SOX2 expression in ovarian cancer (A.) OV2008 and OVCAR3 cells were used for luciferase reporter assay. Luciferase activity was detected 2 days after transfection. (B, C.) The relative expression levels of SOX2 at the protein and mRNA levels in control, mimic and inhibitor transfected groups were determined by immunoblotting and RT-PCR, respectively. (D.) The relative mRNA level of SOX2 in miR-control, mimic and inhibitor transfected CSLCs was determined by RT-PCR, where, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Fig. 4

MiRNA-3163 attenuates the stemness property of the ovarian cancer cells. (A.) Flow cytometry analysis showing percentage of CD44+CD117+ cells in OV2008 and OVCAR3 cell lines following transfection with miRcontrol, mimic and inhibitor. (B.) Spheriod assay and bar diagram showing average spheroids diameter formed in different groups,scale bar=100μm. (C.) Average number of spheroids formed in each microscopic fields with miRcontrol, mimic and inhibitor transfected cells. where, * = P < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.