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

Eleven malignant ovarian tumor tissues and normal ovarian tissues (as listed in Table 1) were selected from the archives of the Department of Obstetrics & Gynecology at Ren Ji Hospital, Shanghai JiaoTong University School of Medicine (Shanghai, China). This study was approved by the Institutional Review Board of Ren Ji Hospital, Shanghai JiaoTong University School of Medicine; and written informed consent was obtained from all patients. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki.

Human ovarian SKOV3, HO8910pm cell lines with high-metastasis potential [30], HO8910, ES-2 and A2780 were obtained from Shanghai Institute of Cell Biology, China Academy of Sciences (Shanghai, China). OVCAR3 and OV90 were obtained from ATCC. SKOV3-ip1 and SKOV3-ip2 cells were selected from SKOV3 cell. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (GIBCO) in a humidified atmosphere of 5% CO₂ at 37°C [31].

To select a highly invasive subpopulation, SKOV3 cells were seeded in a corning 8 μm-pore transwell with Matrigel (BD Biosciences). After 24 hours, cells that had invaded to the other side of the transwell membrane were collected, expanded and then re-seeded into another Matrigel coated transwell. Such selection rounds for highly invasive cells were repeated two times.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Single-stranded cDNA was synthesized with the PrimeScript Reagent Kit (Promega, Madison, WI, USA). Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) on an ABI 7300HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). Expression data were normalized to the internal control (U6) and the relative expression levels were evaluated using the Ct method. Primers for RT–PCR are listed in Table 2. For the protein expression analyses, standard Western blotting was carried out and the antibodies used were SphK1 (H00008877-M01, Abnova, Taiwan) and β-Actin (CP01, Calbiochem, MA, USA).

MiR-124 and the negative control were synthesized by Genepharma (Shanghai, China). Three siRNA duplex oligonucleotides fragments against SphK1 gene were synthesized by Ribobio (Guangzhou, China). Oligonucleotides were transfected with Lipofectamine 2000 reagent (Invitrogen, Paisley, Scotland, UK) at a concentration of 100 nM. For proliferation assays, cells were trypsinized 24 h after transfection. For migration, invasion, and western blot assays, cells were collected 48 h after transfection, as described previously.

For monolayer wounding assays, cells were plated in BD Falcon 24-well tissue culture plates after transfection and allowed to attach overnight. After serum deprivation for 12 h, confluent monolayers were scratched using a 10-μl pipette tip and washed once with serum-free medium. Twenty-four hours later, migration was assessed microscopically, and quantified using Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan).

Cell migration was determined in Corning transwell insert chambers as described previously [31]. Cells suspended in 200-μl serum-free 1640 medium were placed into the upper chamber of the insert with or without Matrigel. After 24 h of incubation, cells remaining on the upper membrane were carefully removed. Cells that had migrated through the membrane were fixed with methanol and stained with 0.1% crystal violet (Biotime, China), imaged and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan).

HO8910pm and SKOV3-ip cells were plated into 24-well plates until 70% confluence before transfection. 100 ng wild-type or mutant SphK1 3’-UTR psiCHECK-2 plasmid (Promega, Madison, WI) was transiently co-transfected with 60 pmol miR-124 mimics or NC into HO8910pm and SKOV3-ip cells. Cell lysates were harvested 24 h after transfection and then firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) on a Berthold AutoLumat LB9507 rack luminometer. Renilla luciferase activities were normalized to firefly luciferase activities to control for transfection efficiency.

Data were expressed as the mean ± SD of at least three independent experiments. Group differences were compared using one-way ANOVA or two-tailed Student’s T-test from SPSS version 19.0 software (SPSS, Chicago, IL, USA). p value <0.05 was considered to be statistically significant.

To determine the expression level of miR-124 in ovarian cancer progression, we first compared the expression levels between 13 clinical tumor samples and 2 normal ovarian tissues by stem-loop qRT-PCR. As shown in (Figure 1A and Table 1), the results showed that expression of miR-124 was decreased in 13 cases of ovarian cancer samples (p < 0.001), compared to normal ovarian tissues. Furthermore, we examined the expression of miR-124 between five paired metastatic and primary ovarian cancer tissues. Interestingly, we observed that the expression level of miR-124 was lower in metastatic tissues than in primary ovarian cancer samples (p < 0.05) (Figure 1B).

We further detected the abundance of miR-124 in nine ovarian cancer cell lines. Compared with HOSE (Pricells, Wuhan, China), a cell derived from human ovary surface epithelial cells, the expression of miR-124 evaluated by real time PCR in the above nine ovarian cancer cell lines were significantly reduced at different extent (Figure 1C), much lower in SKOV3-ip (Additional file 1) and HO8910pm [31] cells in particular. These results suggest that the expression of miR-124 is significantly decreased in human ovarian cancer specimens and cell lines, which may be involved in EOC metastasis.

To investigate the functional role of miR-124 in epithelial ovarian cancer (EOC), we restored miR-124 expression in SKOV3-ip and HO8910pm cells, which shows the lowest expression of miR-124 in the nine ovarian cancer cell lines, by transient transfection with miR-124 mimics or negative control (NC). To verify the overexpression of miR-124, stem-loop qRT-PCR was performed and the results showed significantly increased expression of miR-124 in SKOV3-ip cells (Additional file 2A). In additionally, proliferation rate of cells post miR-124 transfection appeared no change (Additional file 3A, B). Given that SKOV3-ip and HO8910pm are highly metastatic cells, we then investigated the effect of miR-124 on their migration and invasion ability. The wound healing assay as well as migration Transwell assay indicated the ectopic expression of miR-124 can significantly inhibit cell migration compared to control group (Figure 2A, B). The same results were also observed in other two ovarian cancer cell lines, OV90 and OVCAR3 (Additional file 3C, D). Furthermore, invasion capability of SKOV3-ip and HO8910pm cells transfected with NC or miR-124 mimics were evaluated by Matrigel Invasion assay. As expected, the invasion ability of these two cells was decreased markedly as a result of overexpression of miR-124 (Figure 2C).

It is well known that miRNAs regulate gene expression post transcriptionally by binding to the 3’-UTR of mRNAs. To verify the target involved in progression of ovarian cancer, we searched putative target genes via miRanda and TargetScan and we focused on SphK1 because of its rank and function associated with migration and invasion, particularly, the ectopic expression of miR-124 substantially decreased the expression of SphK1 in both SKOV3-ip and HO8910pm ovarian cancer cells assessed by western blot assay (Figure 2D and Additional file 2C), although real-time PCR analysis did not show obvious changes in mRNA expression of SphK1 (Additional file 2B). Besides, we also found that the ovarian cancer samples and metastatic ovarian cancer tissues showed a high level expression of SphK1 (Additional file 4A, B). Similarly, a much higher level expression of SphK1 is also observed in the SKOV3-ip and HO8910pm cells with high-metastasis potential showed (Additional file 4C, D). Collectively, the reduced miR-124 expression and overexpressed SphK1 was probably associated in ovarian cancer tissues and cells, which indicates that SphK1 was a direct target of miR-124.

In order to confirm that miR-124 directly targets SphK1, luciferase assay was performed. SKOV3-ip and HO8910pm cells were transfected with wild-type full length 3’-UTR of Sphk1 which was cloned into the psiCHECK™2 Vector as a control and mutant vector contained 4 mutated bases on only one biding site on SphK1-3’-UTR (Figure 2E and Additional file 2D upper panel), as well as miR-124 or NC. Overexpression of miR-124 significantly suppressed the luciferase activity of reporter genes containing 3’UTR of SphK1 compared with controls but partially rescued when the binding site was mutated (Figure 2F and Additional file 2D lower panel). The results are consistent with previous research [23], indicating that SphK1 is a predicted target of miR-124, and the inhibitory effect of miR-124 is due to direct interaction with the putative binding site of the 3’-UTR of SphK1.

To investigate whether MiR-124 has its inhibitory effect on migration and invasion of ovarian cancer through its target gene SphK1, we next determined to silence SphK1 and evaluated its expression by western blot in SKOV3-ip and HO8910pm cells. The silencing of SphK1 was confirmed by western blot (Figure 3A). In addition, knock-down of SphK1 did not affect the proliferation rate of SKOV3-ip and HO8910pm cells transfected with siRNA against SphK1 (Additional file 5A, B). Subsequently, we assayed migration and invasion ability of these two cells transfected with pooled siRNA fragments, migration and invasion were suppressed (Figure 3B, C and D). These results suggest that SphK1 participates in the motility of ovarian cancer cells.

In order to investigate the contribution of SphK1 to cellular migration and invasion, we ectopically expressed SphK1 together with miR-124 in SKOV3-ip cells to evaluate whether this may overcome the suppressive effect of miR-124 on cell migration and invasion. The SKOV3-ip cells were co-transfected with NC or miR-124 together with pCDNA3.1 (−)-vector or pCDNA3.1 (−)-SphK1 for 48 h. The expression of SphK1 recovered after SphK1 transfection (Figure 4A). Migration and invasion assays showed that enforced expression of SphK1 reversed miR-124-induced inhibition of migration and invasion (Figure 4B, C). Collectively, our results indicate that miR-124 participates in SphK1-mediated migration and invasion of EOC cells, suggesting that SphK1 is a functional mediator of miR-124 in EOC metastasis.