Effect of siRNA-silencing of SALL2 gene on growth, migration and invasion of human ovarian carcinoma A2780 cells

Seven OC cell lines (COC1, HO8910, OVCAR-3, HEY, CAOV3, A2780, and SKOV3; the catalogue numbers of these cell lines are 3111C0001CCC000368™, 3131C0001000700024™, 3131C0001000700108™, 3131C0001000700111™, 3111C0001CCC000339™, 3111C0002000000075™ and 3131C0001000700107™, respectively.) were obtained from Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China). COC1 and CAOV3 were maintained in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA); HO8910 was maintained in Dulbecco’s modified Eagle medium high glucose (DMEM/HG) containing 10% FBS. The other cell lines were maintained in DMEM/F12 containing 10% FBS.

The cells were divided into three groups: Blank control group (untreated), Scramble group (transfected with nontarget siRNAs), and SALL2 siRNA group (transfected with SALL2 siRNAs). The A2780 cells were transfected with three SALL2 siRNAs, namely siRNA1 duplexes (sense: 5′-CCAGCAGUGGCUUGCCUUAUGGUAU-3′; antisense: 3′-GGAAGGAGAUGGACAGUAAUGAGAA-5′), siRNA2 duplexes (sense: 5′-AUACCAUAAGGCAAGCCACUGCUGG-3; antisense: 3′-CAACAACUCUUCGGCCUCCUCUGAA-5′), and siRNA3 duplexes (sense: 5′-UUCUCAUUACUFUCCAUCUCCUCCUCCC-3; antisense: 3′-UUCAGAGGAGGCCGAAGAGUUGUUG-5′). Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) (9 μl) was added to Opti-MEM (250 μl) and mixed for 5 min. Each siRNA (Invitrogen, Carlsbad, CA, USA) (3 μl) and Opti-MEM (250 μl) were mixed. The diluted Lipofectamine and siRNA were mixed for 15 min. The reagents were added into six-well plates, in which A2780 cells were seeded (5 × 10⁵ cells/well) for 24 h. The cells in the Scramble group were treated with Stealth™ RNAi Negative Control Duplex (Invitrogen). The positive control cells were treated with BLOCK-iTTM Alexa Fluor® Red Fluorescent Oligo. The transiently transfected cells were assayed through quantitative real-time PCR (qRT-PCR) and Western blot analysis after transfection for 48 h.

The transfected A2780 cells at a density of 1 × 10⁶ cells/mL were cultured on 35-mm glass-based culture dishes containing DMEM with 10% FBS at 37 °C for 24 h under 5% CO₂. The cells were fixed and permeabilized, followed by staining overnight with mouse anti-Human SALL2 (1:50) mAb in a humidified box at 4 °C. The secondary CY5-conjugated goat anti-mouse antibody (1:100) was subsequently added and incubated for 1 h at room temperature. The cells were washed in cold PBS two times for 3 min and then analysed through CLSM (Olympus, IX71, Tokyo, Japan). The nuclei of the cells were stained with Hoechst 33,258 (Amresco, USA). Isotype controls (Invitrogen, Carlsbad, CA, USA)were used in each experiment.

At 48 h post transfection of the A2780 cells with siRNA, 4 × 10³ cells/mL were introduced into a 96-well plate at 100 μl/well. The cells were incubated at 37 °C under 5% CO₂. They were subsequently incubated for an additional 2 h with 10 μl CCK-8 (Dojindo, Kumamoto, Japan) for 24, 48, and 72 h. The absorbance at 450 nm was measured using a microplate reader (Tecan M200 PRO, Switzerland). Cell proliferation ability was determined as follows: cell proliferation ability = AV (Absorbance value)/0 h AV.

At 48 h post transfection of the A2780 cells with siRNA, 1 × 10⁵ cells/mL were introduced into a 24-well plate at 500 μl/well. The cells were cultured at 37 °C for 24 h under 5% CO₂ according to the instruction manual of the Annexin V-FITC/propidium iodide (PI) Cell Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China). The cells were subsequently treated with 0.5 μg/ml cisplatin (Hansoh Pharmaceutical Co., Ltd., Lianyungang, China) for 18 h, and then digested with 0.25% trypsin (without EDTA), washed with PBS, centrifuged at 2000 rpm for 5 min, and collected. The collected cells were suspended in 500 μl of binding buffer to which 5 μl of Annexin V-FITC and 5 μl of PI were added. The mixture was incubated in the dark for 15 min at room temperature and analysed through flow cytometry (FCM, FACS Aria III, Becton Dickinson, USA).

At 48 h post transfection of the A2780 cells with siRNA, 2.5 × 10⁵ cells/mL were introduced into a 6-well plate at 2 ml/well. All adherent and floating cells were harvested, fixed gently in 70% ethanol overnight at 4 °C, and resuspended in 500 μl of PBS containing 25 μl of PI (20×) and 100 μl of RNase A (50×). After incubation at 37 °C in the dark for 30 min, the cells were analysed by FCM. Data were analysed using the Cell Quest software (BD Biosciences, San Jose, CA, USA).

An xCELLigence Real-Time Cell Analyzer (RTCA) DP (ACEA Biosciences, San Diego, CA, USA) was used in this study. Cell-culture media (100 μl) was added in the lower chamber of the system at room temperature. A CIM-plate 16 was connected to the system. The cell-culture incubator was examined to ensure appropriate electrical contacts, and the background impedance was measured. At 48 h post transfection with siRNA, the cells were resuspended in a cell-culture medium, and the cell density was adjusted to 10⁵ cells/well. Complete medium (165 μl) and serum-free medium (30 μl) were added in the upper and lower chambers, respectively. After 30-min incubation at room temperature, CIM-plate 16 was placed in a cell-culture incubator. Cell migration was monitored every hour for 76 h by using the incorporated sensor electrode arrays of the CIM-plate 16. Electrical impedance was measured using the RTCA-integrated software of the xCELLigence system as a dimensionless parameter termed CI.

Cell invasion was assessed using 24-well inserts (BD Biosciences Cat. No. 354480) with 8-μm pores according to manufacturer instructions. In brief, 48 h post transfection with siRNA, 1 × 10⁵ cells were seeded into the upper chamber with (to measure invasion) or without (to measure migration) the matrigel layer and were allowed to invade the lower reservoir containing 20% FBS at 37 °C for 24 h. After 48 h, the noninvading cells in the upper surface of the filters were removed using a cotton swab; the cells were fixed in 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet for 5 min. The cells in five visual fields that passed through the membrane were counted as invasive cells. All the cells were counted using a microscope at ×200 magnification.

The relative expression levels of SALL2 and p21 were measured through qRT-PCR. The total RNA was extracted using the Trizol reagent (Takara, Dalian, China) and then reverse-transcribed into cDNA by using a Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Canada). The forward and reverse primers used for SALL2 DNA amplification were 5′-CATCCTCAGCCTCTTCTGGA-3′ and 5′-TGCAGAGTGACAGCATTGG-3′, respectively; the forward and reverse primers used for p21 DNA amplification were 5′-CAGGTCCACATGGTCTTCCT-3′ and 5′-TGCCCAAGCTCTACCTTCC-3′. The housekeeping gene GAPDH was used as an internal control, and the primers used for GAPDH were 5′-GCGGGGCTCTCCAGAACATCAT-3′ and 5′-CCAGCCCCAGCGTCAAAGGTG-3′. The qPCR reaction system consisted of 10 μl of FastStart Universal SYBR Green Master Kit (Roche Diagnostics, Basel, Switzerland), 0.5 μl of forward and reverse primers (2.5 μM), respectively, 1 μl of cDNA, and 8 μl of ddH₂O. Probe amplification was performed as follows: 15 s at 95 °C; 45 cycles at 95 °C for 5 s and at 60 °C for 30 s. The relative mRNA expression levels of SALL2 and p21 were normalised against GAPDH by using the comparative △△Ct method. The relative fold change of gene was calculated using the 2^−△△Ct formula.

At 48 h after transfection, the cells were collected and lysed using the RIPA lysis buffer (Beyotime, China). A BCA Kit (Pierce, Rockford, IL, USA) was used to estimate the protein concentration. Protein samples (30 μg/lane) were diluted in 5× SDS-PAGE sample-loading buffer and electrophoretically separated on a 10% SDS-PAGE gel. After the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), the membranes were blocked with 5% nonfat skimmed milk for 2 h at room temperature and then incubated overnight with primary antibodies at 4 °C. After being washed for 5 min three times, the membranes were incubated with suitable secondary antibodies (1:6000; horseradish peroxidase-labelled) for 1 h at room temperature. Signals were detected using ECL reagents (Pierce, USA). ImageJ software was used to measure the grey value of molecular bands. The following antibodies were used: mouse anti-human SALL2 monoclonal antibody (mAb) (Santa Cruz, CA, USA), rabbit anti-human MMP-2 mAb (San Eagle BioTECH, Wuhan, China), rabbit anti-human MMP-9 mAb (San Eagle BioTECH, Wuhan, China), rabbit anti-human Akt mAb (San Eagle BioTECH, Wuhan, China), rabbit anti-human p-Akt mAb (Bioworld TECH, Nanjing, China), mouse anti-human GAPDH mAb (San Eagle BioTECH, Wuhan, China), rabbit anti-human GAPDH polyoclonal antibody (Goodhere BioTECH, Hangzhou, China) HRP-conjugated goat anti-mouse antibody, and HRP-conjugated goat anti-rabbit antibody (both from Zhongshanjinqiao BioTECH, Beijing, China).

LY294002 (Cell Signalling Technology, Boston, USA), a PI3K inhibitor, was prepared in DMSO from a stock concentration of 50 mM. The A2780 cells were detached by trypsinization and washed two times with PBS. At 48 h post transfection with siRNA, the transfected cells were cultured in 6-well plates (5 × 10⁵ cells/well) for 24 h and then incubated with 50 μM of DMSO or LY294002 for 30 min. The cell pellets were subsequently collected for Western blot analysis. The following antibodies were used: mouse anti-human Akt mAb (San Eagle BioTECH, Wuhan, China) and rabbit anti-human p-Akt mAb (San Eagle BioTECH, Wuhan, China). For the transwell assay, transfected cells were cultured overnight in 24-well plates (1 × 10⁵ cells/well). Either DMSO (50 μM) or LY294002 (50 μM) was added to the cells for 30 min and then the cells were collected for the transwell experiment.

All data were analysed using SPSS version 17.0 for Windows. Continuous variables were expressed as means ± standard deviations. Differences between the Scramble and SALL2 siRNA groups were assessed using the Student’s t-test; comparisons involving three or more groups were performed using one-way ANOVA. At least three repetitions (n = 3) were performed on the data in each group. P < 0.05 was considered statistically significant.

RT-PCR and Western blot analysis were used to determine SALL2 expression in seven human OC cell lines (A2780, COC1, HO8910, SKOV3, CAOV3, HEY, and OVCAR3). The results indicated that gene and protein expression of SALL2 were detected in the SKOV3, A2780, COC1, CAOV3, OVCAR3, and HO8910 cells. The protein expression levels of SALL2 were the highest in the A2780 cells; hence, we selected the A2780 cells for subsequent experiments (Fig. 1).

In the A2780 cells, siRNAs were used to downregulate SALL2 expression. The results indicated that the transfection efficiency of the positive control was 81.2%, indicating that SALL2 siRNAs were successfully transfected into the A2780 cells (Fig. 2a and b). SALL2 expression was significantly lower in the siRNA2 group than in the siRNA1, siRNA3, Scramble, and Blank control groups (P < 0.01). Moreover, SALL2 expression was lower in the siRNA3 group than in the siRNA1, Scramble, and Blank control groups (P < 0.05), and SALL2 expression did not significantly differ among the siRNA1, Scramble, and Blank control groups. Because siRNA2 efficiently silenced SALL2, it was selected for subsequent silencing experiments (Fig. 2c and d). SALL2 siRNAs transfection regulated SALL2 mRNA levels at 24 h and 72 h. (Additional file 1: Figure S1). SALL2 expression in the A2780 cells transfected with siRNA was further confirmed by CLSM. The results indicated that in the Blank control and Scramble group, SALL2 was detected both in the cytoplasm and nucleus. By contrast, SALL2 was not detected in the cytoplasm or nucleus of the cells in the siRNA2 group (Fig. 2e).

To determine the role of SALL2 in the growth of OC cells, we analysed the proliferative ability, apoptosis extent, and cell cycle status of the A2780 cells transfected with SALL2 siRNA. At 24, 48, and 72 h, the A2780 cells transfected with SALL2 siRNA exhibited higher levels of cell viability than did the cells in the Scramble group (P < 0.05) (Fig. 3a). The A2780 cells transfected with SALL2 siRNA for 48 h exhibited a lower extent of apoptosis than did the cells of the Scramble group (P < 0.05) (Fig. 3b; 22.3% early apoptotic cells and 4.5% late apoptotic cells in Scramble group vs. 16.2% and 2.6% in A2780 cells at 48 h after siRNA infection). The number of A2780 cells transfected with SALL2 siRNA in the G0/G1 phase was lower than that of the cells in the Scramble group (P < 0.05) for 48 h (Fig. 3c; 56.94% G0/G1 phase cells in Scramble group vs. 48.87% in A2780 cells at 48 h after siRNA infection). Despite these disparities, no significant differences were observed between the Scramble and Blank control groups in terms of proliferation, cell cycle, and apoptosis extent (P > 0.05). These results indicated that SALL2 downregulation promoted tumour cell proliferation and cell cycle progression and reduced cell apoptosis.

Migration of cancer cells is one of the key factors responsible for cancer metastasis [14]. The results of the RT-CIM migration assay indicated that the cell index significantly increased in cells transfected with SALL2 siRNA (P < 0.05) compared with the cells in the Scramble group (Fig. 4a). We performed the transwell assay to investigate the invasion capacity of the transfected A2780 cells. The number of invasive cells was significantly higher in the SALL2 siRNA group (P < 0.05) compared with that in the Scramble group (Fig. 4b). No significant differences were observed between the Scramble and Blank control groups (P > 0.05). These results indicate that SALL2 downregulation promotes tumour cell migration and invasion, resulting in OC cell migration and invasion.

To investigate the mechanism of the effects of SALL2 expression on growth of the A2780 cells, we detected p21 by using qRT-PCR. The results revealed that the mRNA expression of p21 was significantly lower in the SALL2 siRNA group than in the Scramble group (P < 0.05), thereby indicating that SALL2 silencing downregulates p21 mRNA expression (Fig. 5). Considering that p21 gene is the downstream target of p53, it constitutes the checkpoint of the G1 phase of the cell cycle to reduce the replication and accumulation of damaged DNA; thus, it inhibits the growth of osteosarcoma cells [15]. We speculate that the promotion of A2780 cell growth by SALL2 downregulation is associated with p21.

To identify the mechanism underlying the promotion of the migration of and invasion by A2780 cells through SALL2 downregulation, we investigated the MMP2 and MMP9 proteins through Western blot analysis. The results indicated that protein expression of MMP2 and MMP9 was higher in the SALL2 siRNA group than in the Scramble group (P < 0.05), thereby indicating that silencing SALL2 upregulates MMP2 and MMP9 protein expression (Fig. 6A).

To investigate whether the migration of and invasion by A2780 cells are correlated with activation of the PI3K/Akt signalling pathway, we detected the Akt and p-Akt proteins through Western blot analysis. The results indicated that protein expression of p-Akt was higher in the SALL2 siRNA group than in the Scramble group (P < 0.05). However, the expression level of Akt did not change considerably (Fig. 6B).

In the SALL2 siRNA group, the cells treated with LY294002 exhibited lower expression levels of p-Akt and a lower number of migrating and invading cells than did the cells treated with DMSO (P < 0.05). The total Akt protein levels did not differ between the two treatments (Fig. 6c and d). In the Scramble group, the expression of p-Akt and Akt and migration of and invasion by the A2780 cells were not affected by DMSO or LY294002 treatment (P > 0.05).

These results suggest that SALL2 downregulation elevates MMP2 and MMP9 protein expression levels and activates the PI3K/Akt pathway, thereby accelerating migration and invasion of OC cells.