| Objective:To investigate the effects of sphingosine-1-phosphate receptor 1(S1PR1)on the expression of cytokines(IL-6,IL-8,VEGF),angiogenesis and macrophage phenotype in the microenvironment of esophageal squamous cell carcinoma(ESCC)cell line Eca109,and screen differentially expressing genes(DEGs)and signaling pathways related to sphingosine 1-phosphate(S1P)/S1PR1 pathway in ESCC cells,so as to provide a scientific basis for the pathogenesis,diagnosis,treatment and prognosis of esophageal cancer.Methods:(1)The expression of sphingosine kinase 1(SPHK1)and S1PR1 in ESCC cell lines Eca109,TE-1 and KYSE150 was detected by quantitative reverse transcript-PCR(qRT-PCR).(2)S1PR1-EGFP and Control-EGFP were respectively transfected into Eca109 cells by lipofectamineTM 2000 for transient expression.qRT-PCR was used to detected the expression level of S1 PR 1 mRNA in Eca 109 cells,and the expression levels of cytokines IL-6,IL-8 and VEGF in conditioned medium(CM)were detected by ELISA.(3)S1PR1-EGFP and Control-EGFP were transfected into Eca109 cells respectively.24-48h and 48h CM was applied to endothelial EA.hy926 cell to observe tube formation.(4)THP-1 cells were differentiated into macrophages using PMA.Macrophages were treated with 24-48h and 48h CM,and qRT-PCR was used to detect the expression levels of macrophage markers such as CD68,CD163,IL-1β,iNOS to evaluate the phenotypes of macrophages.(5)RNA sequencing(RNA-seq)was employed to screen out differentially expressed genes between 24-48h S1PR1-EGFP and Control-EGFP-Eca109 cells and conduct bioinformatics analysis.(6)The role of differentially expressed gene GALR2 in the migration of Eca109 cells induced by S1PR1 was examined by siRNA and migration experiments.(7)The effects of differentially expressed genes C1s on the migration and proliferation of TE-1 cells were detected by siRNA,migration experiments,and CCK-8 proliferation experiments.Results:(1)qRT-PCR was used to detect the expression levels of SPHK1 and S1PR1 mRNA in Eca 109,TE-1,and KYSE150 cells.The results showed that the expression level of SPHK1 in Eca109 were higher than that in TE-1 and KYSE150(P<0.01),and the expression levels of S1PR1 in TE-1 were much higher than that of Eca109 and KYSE150(P<0.01).(2)qRT-PCR was used to detect the expression level of S1PR1 in Eca109 cells transfected with S1PR1-EGFP and Control-EGFP.The results showed that the expression levels of S1PR1in the S1PR1-EGFP group were significantly higher than those in the Control-EGFP group(P<0.01.(3)The expression levels of IL-6,IL-8,VEGF were detected by ELISA.The results showed that except for the IL-8 of 48h CM,the expression levels oflL-6,IL-8,VEGF in the S1PR1-EGFP group were higher than those in the Control-EGFP group(P<0.01 or P<0.05).(4)The results of tube formation assay showed that upregulation of S1PR1 may lead to an increase in the capacity of angiogenesis(P<0.01.(5)Macrophages were treated with 24-48h and 48h CM.The results showed that macrophages cultured in CM from overexpressing S1PR1 for 24-48h Eca 109 cells had a lower expression of CD 163 than the control cells(P<0.05).However,macrophages cultured in CM from overexpressing S1PR1 for 48h Eca 109 cells had a higher expression of CD 163(P<0.05)and a lower expression of IL-1β than the control cells(P<0.01).(6)RNA-seq results showed that 574 differentially expressed genes were screened in S1 PR 1-overexpressing Eca109 cells,of which 344 genes were up-regulated and 230 genes were down-regulated.GO analysis showed that differentially expressed genes were involved in the regulation of postsynaptic membrane potential,positive regulation of chronic inflammatory response,complement activation,regulation of ion transport,and so on.KEGG analysis showed that differentially expressed genes were mainly involved in arachidonic acid metabolism,complement and coagulation cascades and staphylococcus aureus infection signaling pathways.PPI analysis showed that the first node was GALR2.(7)Migration experiment was used to detect the migration ability of Eca109 cells after co-transfected with Control-EGFP+NC siRNA,S1PR1-EGFP+NC siRNA,S1PR1-EGFP+GALR2 siRNA.The results showed that the cell migration rate in S1PR1-EGFP+GALR2 siRNA group was significantly lower than that in S1PR1-EGFP+NC siRNA group(P<0.01).(8)Migration experiment was used to detect the migration ability of TE-1 cells after transfection with Lipo,NC siRNA,C1s siRNA-1,and C1s siRNA-2.The results showed that the cell migration rate in C1s siRNA-1 group was lower than that in NC siRNA group(P<0.01).The CCK-8 experiment was used to detect the proliferation of TE-1 cells after transfection with Lipo,NC siRNA,C1s siRNA-1,and C1s siRNA-2.The results showed that the cell proliferation of C1s siRNA-1 and C1s siRNA-2 group was lower than that of NC siRNA group after 24 hours of transfection(P<0.05).Conclusion:(1)Overexpression of S1 PR 1 in Eca 109 cells promotes the secretion of IL-6,IL-8 and VEGF.(2)The CM of S1 PR 1-overexpressing Eca 109 cells promotes angiogenesis.(3)The CM of S1PR1-overexpressing Eca109 cells induces macrophages to show a mixed M1/M2 phenotype and has a trend to polarize macrophages into M2 phenotype.(4)Differentially expressed genes related to S1P/S1PR1 pathway were identified in S1PR1-overexpressing Eca 109 cells.Both GO and KEGG enrichment analysis showed that some differentially expressed genes(such as C1s)were related to the complement system,and PPI analysis showed that the first node was GALR2.(5)S1PR1 may induce the migration of Eca 109 cells through the mediation of GALR2.(6)TE-1 cells have a high expression of S1PR1 and C1s,and knockdown of C1s expression inhibit the migration and proliferation of TE-1 cells. |
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