| Objective:To explore the role of SOS1 in malignant behaviors of epithelial ovarian cancer (EOC) cells Hey with high metastatic potential.Methods:1. In vitro experiments:the matrix-cell adhesion was tested by matrix adhesion assay in Hey cells with SOS1 deficiency (Hey-SOSli), as compared to negative control (Hey-NT) and blank control (Hey-WT); the changes of cellular morphology, actin cytoskeleton, pseudopodia and cell arrangement in three groups were observed by immunofluorescence for phalloidine; Invasive ability of tumor cells was evaluated by Matrigel transwell, intravasation and extravasation assay; MTT and plate clone formation assay were performed to measure proliferation ability; Cellular apoptosis and cycle were evaluated by flow cytometry measurement; DNA damage was assessed by Comet assay; 2. In vivo experiments: Subcutaneous xenograft and intraperitoneal implantation metastasis model in nude mice were established to observe the tumorigenesis and metastasis potency of Hey cells.3 Exploring the underlying mechanism:Real time PCR and Western Blot were used to detect the RNA and proteins probably involved in Sosl function; The nuclear translocation of snail was assessed through immunofluorescence.Results:Firstly, compared with Hey-WT and Hey-NT cells, Hey-SOSli cells showed decreased polarity, disorder in cytoskeleton arrangement; Numbers of transwell migrated cells (90.00±2.45 vs.274.67±8.18 vs.254.00±2.16, P＜0.01), invaded cells (24.00±1.25 vs.112.33±2.54 vs.102.00±8.94, P<0.01), intravasated cells (13.00±1.63 vs 27.33±1.24 vs.24.00±2.16, P<0.05) and extravasated cells(9.00±2.16 vs.27.22±1.69 vs.32.33±1.05, P<0.05) were decreased significantly. Furthermore, the adhesiveness to ECM matrix was significantly weakened (0.31±0.10 vs.0.72±0.10 vs.0.69±0.13, P<0.05); and decreased growth speed and less clones were observed (0.13±0.10 vs.0.36±0.02 vs.0.41±0.06, P<0.05). However, no significant difference could be found of the proportion of apoptotic cells (3.52%±0.05%vs.5.64%±2.37% vs.3.11%±0.55%, P>0.05) and the DNA damage (45.86±12.81vs.47.24±10.48 vs.43.58±14.01, P>0.05) among the three groups. Hey-NT cells and Hey-SOSli cells were employed to establish peritoneal dissemination model and subcutaneous model in nude mice. The volume and weight of xenograft of Hey-SOSli cells (2.18±1.46cm3 and 2.31±0.98g) were lower than the Hey-NT cells xenograft (0.74±0.16cm3, P<0.05 and 0.73±0.20g, P<0.05) at the 35th day after implantation. In addation, Hey-SOSli cells formed less implantation metastatic foci in the abdominal cavity than Hey-NT cells(83.33±17.06 vs.49.33±5.65, P<0.05), especially on the intestine (58.67±10.53vs.33.0±4.32, P＜0.05) and diaphragm (16.67±3.21 vs.10.33±0.58, P<0.05) at the 5th week after the tumor cells were injected intraperitoneally. SOS1 knockdown in Hey cells resulted in increased E-cadherin and decreased vimentin, N-cadherin, MMP2 and MMP9, together with reduced Snail and activation of NF-kB pathway.Conclusion:SOS1 might induce EMT through activating NF-kB and the transcriptive activity of Snail, which thus regulates the cytoskeleton reprogramming and cell motiligy of HEY, one EOC cells with high metastatic potential. |
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