Effects Of S1PR1 On Cell Cycle And Migration Of Human Esophageal Squamous Cell Carcinoma Cell Line Eca109

Objective: To investigate the effects of sphingosine-1-phosphate receptor 1(S1PR1) on cell cycle, cell cycle related molecules p21CIP1/WAF1(p21) and p27KIP1(p27), and migration in human esophageal squamous cell carcinoma(ESCC) cell line Eca109, and provide new ideas for the pathogenesis, tumor diagnosis, treatment and prognosis of esophageal cancer.Methods:(1) S1PR1-EGFP vector or Control-EGFP vector was transfected into Eca109 cells by the LipofectamineTM 2000. The expression and localization of S1PR1-EGFP and Control-EGFP were analyzed by confocal laser scanning microscope(CLSM) at 12 h, 24 h and 48 h post-transfection.(2) The cell cycle of Eca109 cells was detected by flow cytometry at 12 h and 48 h post-transfection.(3) The m RNA expression of p21 and p27 in Eca109 cells was detected by real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR) at 48 h post-transfection.(4) The protein expression of p21 and p27 in Eca109 cells was detected by Western blot at 48 h post-transfection.(5) The migration capability of Eca109 cells was detected by Transwell at 48 h post-transfection.Results:(1) The green fluorescence of S1PR1-EGFP was predominantly localized at the plasma membrance of Eca109 cells at 12 h post-transfection. The green fluorescence of S1PR1-EGFP was predominantly localized at the plasma membrance and cytoplasm of Eca109 cells at 24 h post-transfection. The green fluorescence of S1PR1-EGFP was predominantly localized at the cytoplasm of Eca109 cells at 48 h post-transfection. At the all three time points, Control-EGFP was localized at the cytoplasm and nucleus.(2) The proportions of G1 phase cells in S1PR1-EGFP-transfected Eca109 cells and Control-EGFP-transfected Eca109 cells were 67.75 ± 2.80% and 46.23 + 4.37% at 12 h post-transfection. The proportions of G1 phase cells in S1PR1-EGFP-transfected Eca109 cells and Control-EGFP-transfected Eca109 cells were 30.64±1.91% and 42.99±3.53% at 48 h post-transfection(P<0.01).(3) The results of q RT-PCR showed that, the m RNA expression level of p21 and p27 in S1PR1-EGFP group was significantly lower than that in Control-EGFP group at 48 h post-transfection(P<0.01).(4) The results of Western blot showed that, the protein expression level of p21 and p27 in S1PR1-EGFP group was significantly lower than that in Control-EGFP group at 48 h post-transfection(P<0.01).(5) Transwell assay results showed that, the migrated number of S1PR1-EGFP-transfected Eca109 cells was more than that of Control-EGFP-transfected Eca109 cells at 48 h post-transfection(P<0.01).Conclusions:(1) At 48 h post-transfection, the green fluorescence of S1PR1-EGFP was predominantly localized at the cytoplasm of Eca109 cells. The result was consistent with immunohistochemistry and immunocytochemistry. Therefore, we choose 48 h as the main point of time.(2) Cytoplasmic S1PR1 can promote Eca109 cells from G1 phase to S phase, enhance the proliferation of Eca109 cells.(3) Cytoplasmic S1PR1 can significantly promote G1/S phase transition, which may be associated with the down-regulation of p21 and p27.(4) Cytoplasmic S1PR1 can promote the migration of Eca109 cells.