MiR-124 inhibits the migration and invasion of ovarian cancer cells by targeting SphK1

Abstract

Background: Epithelial ovarian cancer (EOC) is still a major gynecologic problem with poor 5 year survival rate due to distance metastases, despite routine surgery and chemotherapy. The precise underlying molecular mechanisms that trigger EOC migration and invasion are unclear. Recent studies suggest that the expression of microRNAs is widely dysregulated in ovarian cancer; and that they have evolved into tumorigenic processes, including cell proliferation, apoptosis and motility.

Methods: The expression of miR-124 was assessed in clinical ovarian cancer specimens and cell lines using miRNA qRTPCR. The function of miR-124 on cell migration and invasion was confirmed in vitro through wound healing assay and transwell assay. Luciferase reporter assay was used to confirm target associations.

Results: We showed that miR-124 is down-regulated in ovarian cancer specimens as well as in cell lines; and that low-level expression of miR-124 is much lower in highly metastatic ovarian cancer cells and tissues. Meantime, overexpression of miR-124 dramatically inhibits the motility of ovarian cancer cells in vitro and substantially suppresses the protein expression of SphK1, reported as an invasion and metastasis-related gene in human cancers, whose expression is markedly increased in both ovarian cancer cell lines and clinical samples, particularly in two highly metastasis cells, SKOV3-ip and HO8910pm as well as metastatic ovarian tumor tissues. Furthermore, SphK1 is identified as a direct target of miR-124, and knock-down of SphK1 in ovarian cancer cells, SKOV3-ip and HO8910pm, could mimic the inhibition of migration and invasion by miR-124, while re-introduction of SphK1 abrogates the suppression of motility and invasiveness induced by miR-124 in both cell lines.

Conclusions: Our studies suggest a protective role of miR-124 in inhibition of migration and invasion in the molecular etiology of ovarian cancer, and a potentially novel application of miR-124 in the regulation of migration and invasion in EOC.

Figure 1

Expression of miR-124 in cell lines and tissues of ovarian cancer. (A) Expression of miR-124 in normal human ovarian tissues and ovarian tumors. (B) Comparison of miR-124 expression in five paired primary ovarian tumors and metastatic tissues. (C) The relative expression level of miR-124 in nine ovarian cancer cell lines. HOSE, the human normal ovarian epithelial cell line, was used as a control. Data are presented as means ± SD from three individual experiments. The symbols * and *** represent statistical significance at p < 0.05 and p < 0.001, respectively.

Figure 2

MiR-124 directly targets SphK1 and inhibits the migration and invasion of ovarian cancer cells. (A) Wound healing assay of SKOV3-ip and HO8910pm cells transfected with NC or miR-124 mimics. Representative pictures are shown at 0 and 24 h after the wound was made. (B) Transwell assay of SKOV3-ip and HO8910pm cells transfected with NC or miR-124 mimics. (C) Matrigel invasion assay of SKOV3-ip and HO8910pm cells transfected with miR-124 mimics or NC. (D) Expression of endogenous SphK1 in SKOV3-ip cells at 48 h and 72 h post transfection of miR-124 mimics or NC. β-actin was loaded as an internal control. (E) Diagram of the SphK1-3’-UTR with potential binding-sites for miR-124. (F) Relative luciferase activity of reporters including wild-type or mutant SphK1 3’-UTR co-transfected with NC or miR-124 mimics. ** and *** indicate significant difference at p < 0.01 and p < 0.001, respectively.

Figure 3

SphK1 knockdown results in inhibition of migration and invasion in ovarian cancer cells. (A) SphK1 detection in SKOV3-ip and HO8910pm cells transfected with NC or pooled si-SphK1. (B) Wound healing assay of SKOV3-ip and HO8910pm cells after transfected with NC or pooled si-SphK1. (C) Transwell assay of SKOV3-ip and HO8910pm cells subjected to NC or pooled si-SphK1. (D) Matrigel invasion assay of SKOV3-ip and HO8910pm cells after transfection with NC or pooled si-SphK1. Symbols ** and *** represent significance at p < 0.01 and p < 0.001, respectively.

Figure 4

Expression of SphK1 reversed the miR-124-induced inhibition of cellular migration and invasion. (A) The transfection of pcDNA3.1 (−)–SphK1 restored the expression of Sphk1 in SKOV3-ip cells even with miR-124 co-transfection. (B) Transwell assay and (C) Matrigel invasion assay of SKOV3-ip cells after co-transfection of NC or miR-124 mimics together with either pcDNA3.1 (−)-vector or pcDNA3.1 (−)-SphK1. ***denotes statistical significance at p < 0.001.