| Objective:To investigate the effects of sphingosine-1-phosphate（SIP）/sphingosine-1-phosphate receptor 1（S1PR1）pathway regulating the expression of complement factor B（CFB）on the proliferation and migration of esophageal squamous cell carcinoma（ESCC）cells,and the effects of CFB knockdown on the proliferation,migration and tumor formation of ESCC cell line TE-1,so as to provide scientific basis for the pathogenesis,diagnosis,treatment and prognosis of esophageal cancer.Methods:（1）The expression of S1PR1 and CFB in esophageal squamous cell carcinoma cell lines Eca109,TE-1 and KYSE150 was detected by quantitative reverse transcript-PCR（qRT-PCR）.（2）S1PR1-EGFP or Control-EGFP was transfected into Eca109 cells by lipofectamineTM 2000 for transient expression.qRT-PCR was used to detect the expression levels of CFB mRNA48 h after transfection.（3）TE-1 cells was transfected with CFB siRNA or control NC siRNA,the expression level of CFB mRNA was detected by qRT-PCR and the secretion level of CFB was detected by ELISA.（4）S1PR1-EGFP and CFB siRNA were co-transfected into Eca109 cells with LipofectamineTM 2000.The secretion of CFB was detected by ELISA,and the proliferation and migration of cells were detected by CCK8 and wound healing assay.（5）TE-1 cells was transfected with CFB siRNA or control NC siRNA,CCK8 and wound healing assay were used to detect the proliferation and migration of TE-1 cells.（6）CFB shRNA or Control shRNA was transfected into TE-1 cells by LipofectamineTM 2000,G418 was used to screen stable cell lines.qRT-PCR was used to detect the expression of CFB mRNA.ELISA was used to detect the level of CFB secretion,CCK8 and wound healing assay were used to detect proliferation and migration,and clone formation assay was used to determine the clone formation rate.（7）CFB shRNA or Control shRNA stable cell lines was injected into nude mice respectively,and tumor formation was detected.Results:（1）The expression levels of S1PR1 and CFB mRNA in Eca109,TE-1 and KYSE150 cells were detected by qRT-PCR.The results showed that the expression of S1PR1 mRNA in TE-1 cells was much higher than that in Eca109 and KYSE 150 cells（P<0.001）.The expression of CFB mRNA was the highest in TE-1 cells and low in Eca109 and KYSE 150 cells（P<0.001）.（2）qRT-PCR was used to detect the expression of CFB in Eca109 cells 48 h after transfection.The results showed that compared with the control group,the expression level of CFB mRNA in S1PR1-overexpression Eca109 cells was significantly increased（P<0.01）.（3）The results of qRT-PCR and ELISA showed that the expression of CFB mRNA in TE-1 cells transfected with CFB siRNA1 or CFB siRNA2 was significantly lower than that in the control group（P<0.001,P<0.001）,and CFB protein secretion also decreased（P<0.05,P<0.05）.（4）After co-transfection of S1PR1-EGFP and CFB siRNA into Eca109 cells,the secretion level of CFB protein in S1PR1-EGFP+NC siRNA group was higher than that in the Control-EGFP+NC siRNA group（P<0.01）,while the secretion level of CFB protein in S1PR1-EGFP+CFB siRNA1 group or S1PR1-EGFP+CFB siRNA2 group was lower than that in the S1PR1-EGFP+NC siRNA group（P<0.001,P<0.001）.The results of CCK8 assay showed that the proliferation of S1PR1-EGFP+NC siRNA group was higher than that in the Control-EGFP+NC siRNA group（P<0.001）,while S1PR1-EGFP+CFB siRNAl group or S1PR1-EGFP+CFB siRNA2 group was lower than that in the S1PR1-EGFP+NC siRNA group（P<0.001,P<0.001）.The results of wound healing assay showed that the migration of S1PR1-EGFP+NC siRNA group was higher than that in the Control-EGFP+NC siRNA group（P<0.001）,while S1PR1-EGFP+CFB siRNAl group or S1PR1-EGFP+CFB siRNA2 group was lower than that in the S1PR1-EGFP+NC siRNA group（P<0.001,P<0.001）.（5）The results of CCK8 assay and wound healing assay showed that after transfection of CFB siRNAl or CFB siRNA2,the proliferation of TE-1 cells was inhibited（P<0.01,P<0.001）,and the migration was inhibited（P<0.001,P<0.001）.（6）The results of qRT-PCR and ELISA showed that the expression of CFB mRNA in the CFB shRNA stable cell line was lower than that in the control stable cell line（P<0.001）,the secretion level of CFB protein decreased（P<0.01）.The CCK8 results showed that the proliferation of the CFB shRNA stable cell line was significantly inhibited compared with the control stable cell line（P<0.01）,and the wound healing assay showed that the migration was inhibited（P<0.01）.The results of colony formation assay showed that the clone formation rate of CFB shRNA stable cell line was lower than that in the control stable cell line（P<0.01）.（7）The results of transplanted tumor in nude mice showed that the volume of CFB shRNA stable cell line transplanted tumor in nude mice was significantly smaller than that in the control nude mice（P<0.001）.Conclusion:（1）Among the three kinds of esophageal squamous cell carcinoma cells TE-1,Eca109 and KYSE150,TE-1 cells had the highest expression levels of S1PR1 and CFB mRNA.（2）Overexpression of S1PR1 can up-regulate the expression of CFB in esophageal squamous cell carcinoma Eca109 cells.（3）Overexpression of S1PR1 and knockdown of CFB in Eca109 cells,both proliferation and migration were inhibited compared to Eca109 cells with overexpression of S1PR1.（4）CFB knockdown inhibited the proliferation,migration and clone formation of esophageal squamous cell carcinoma TE-1 cells.（5）The volume of transplanted tumor in nude mice with CFB knockdown TE-1 cell line was significantly smaller than that in the control nude mice. |
