| Objective:To investigate the effect of sphingosine 1-phosphate receptor 1(S1PR1)regulating the expression of complement C1s on the proliferation and migration of esophageal squamous cell carcinoma(ESCC)cells,and the effect of complement C1s on proliferation,migration,cell-matrix adhesion of ESCC cells and growth of xenograft tumors in nude mice,so as to provide scientific basis for the pathogenesis and treatment of esophageal cancer.Methods:1.The C1S mRNA expression of ESCC tissues and adjacent normal tissues(ANTs)was analyzed using NCBI-GEO database.2.The expression level of C1s in human ESCC cell lines TE-1,Eca-109,and KYSE-150 cells was analyzed with Western blot.3.LipofectamineTM 2000 was used as a transfection reagent,and S1PR1-EGFP and Control-EGFP were respectively transfected into Eca-109 cells for transient expression.The expression levels of C1s were detected by RT-qPCR and Western blot.4.LipofectamineTM 2000 was used as a transfection reagent,and S1PR1-EGFP overexpression vector and C1s siRNA were co-transfected into Eca-109 cells.The proliferation and migration of cells were detected by CCK-8 assay and wound healing assay,respectively.5.LipofectamineTM 2000 was used as a transfection reagent,C1s siRNA was transfected intoTE-1 and Eca-109 cells.C1s knockdown TE-1 stable cell lines(TE-1-C1s shRNA)were constructed by plasmid transfection.C1s knockdown Eca-109 stable cell lines(Eca-109-C1s shRNA 1)and C1s overexpression Eca-109 stable cell lines(Eca-109-C1s OE)were constructed by lentivirus infection.RT-qPCR and Western blot were used to detectethe expression levels of C1s.6.The expression level of C1s in the culture supernatant of TE-1,Eca-109,KYSE-150 cells and stable cell lines was detected by enzyme-linked immunosorbent assay(ELISA).7.The effects of C1s on the proliferation,migration,and cell-matrix adhesion of ESCC cells were detected by CCK-8 assay,wound healing assay,and cell-matrix adhesion assay,respectively.8.The expression of matrix metalloproteinases MMP1 and MMP13 in stable cell lines was detected by Western blot.9.C1s knockdown TE-1 stable cell lines(TE-1-C1s shRNA),C1s overexpression Eca-109 stable cell lines(Eca-109-C1s OE)and their respective control cell lines were injected subcutaneously into nude mice to detect their tumor formation.10.The expression of CD34 in the xenograft tumors was detected by immunohistochemistry(IHC),and the formation of tumor microvessels was analyzed.Results:1.The analysis in the NCBI-GEO database showed the expression of C1S mRNA in ESCC tissues was significantly higher than that in ANTs(P<0.001).2.Western blot results showed that C1s was highly expressed in TE-1 cells,but lowly expressed in Eca-109 and KYSE-150 cells.3.RT-qPCR and Western blot were used to detect the expression level of C1s in Eca-109 cells transfected with S1PR1-EGFP.The results showed that compared with the control group,the expression level of C1s in Eca-109 cells overexpressed S1PR1 was significantly increased(P<0.05).4.RT-qPCR and Western blot results showed that after TE-1 and Eca-109 cells were transfected with C1s siRNA1 and C1s siRNA3,respectively,the expression of C1s was significantly lower than that in the control group(P<0.05,P<0.01;P<0.01,P<0.001).Compared with the respective control cell lines,the expression of C1s in TE-1-C1s shRNA1 and Eca-109-C1s shRNA1 cell lines decreased(P<0.001;P<0.001),and in Eca-109-C1s OE cell lines increased(P<0.01).5.Western blot results showed that the activated form of C1s(α chain)was detected in all three ESCC cell lines tested.6.ELISA results showed that the expression level of C1s in all cell culture supernatants was relatively low,including C1s-overexpressing cell culture supernatants.7.After co-transfection of SIPR1-EGFP overexpression vector and C1s siRNA into Eca-109 cells,the results of CCK-8 assay showed that the cell proliferation of S1PR1-EGFP+NC siRNA group increased significantly compared with Control-EGFP+NC siRNA group(P<0.01),while the cell proliferation of S1PR 1-EGFP+C1s siRNA 1 group and S1PR1-EGFP+C1s siRNA3 group decreased significantly compared with S1PR1-EGFP+NC siRNA group(P<0.001;P<0.01).Wound healing assay results showed that the migration rate of S1PR1-EGFP+NC siRNA group increased significantly compared with Control-EGFP+NC siRNA group(P<0.001),while the migration rate of S1PR1-EGFP+C1s siRNA1 group and S1PR1-EGFP+C1s siRNA3 group decreased significantly compared with S1PR1-EGFP+NC siRNA group(P<0.001;P<0.001).8.CCK-8 assay showed the proliferation ability of TE-1 and Eca-109 cells transfected with C1s siRNA1 and C1s siRNA3 decreased significantly compared with NC siRNA group(P<0.05,P<0.001;P<0.01,P<0.01).Similarly,compared with the respective control cell lines,the proliferation ability of TE-1-Cls shRNA1 cells and Eca-109-C1s shRNA1 cells was also inhibited(P<0.001,P<0.01),while the proliferation ability of Eca-109-C1s OE cells was significantly increased(P<0.001).9.Wound healing assay results showed that the migration rate of TE-1 and Eca-109 cells transfected with C1s siRNA1 and C1s siRNA3 was significantly inhibited(P<0.001,P<0.001;P<0.001,P<0.001).Similarly,compared with the respective control cell lines,the migration rate of TE-1-C1s shRNA1 cells and Eca-109-C1s shRNA1 cells were also significantly inhibited(P<0.001,P<0.001),while the migration rate of Eca-109-C1s OE cells was significantly increased(P<0.001).10.Cell-matrix adhesion assay showed that compared with the respective control cell lines,the cell-matrix adhesion ability of TE-1-C1s shRNA1 cells and Eca-109-C1s shRNA1 cells was inhibited(P<0.001,P<0.01),while the cell-matrix adhesion ability of Eca-109-C1s OE cells was enhanced(P<0.001).11.Western blot results showed that the expression of MMP1 and MMP13 was decreased in TE-1 stable cell lines with C1s knockdown.12.The results of the subcutaneous xenograft tumor formation experiment in nude mice showed that compared with the control group,the tumor formation rate of the C1s knockdown group(TE-1-C1s shRNA1)was significantly lower.Compared with the control group,the volume and weight of the xenograft tumor in the C1s overexpression group(Eca-109-C1s OE)were significantly larger(P<0.001,P<0.01).13.IHC results showed that compared with the control group,the microvessels of the xenograft tumors in the C1s overexpression group were more abundant(P<0.01).Conclusion:The expression of C1S mRNA in ESCC tissues was significantly higher than that in ANTs.S1PR1 inducing the expression of C1s promoted the proliferation and migration of ESCC cells.C1s knockdown inhibited the proliferation,migration,and cell-matrix adhesion of human ESCC cell lines,while C1s overexpression had the opposite effects.C1s knockdown suppressed the tumor formation rate of TE-1 cells in nude mice,and C1s overexpression significantly promoted the growth of Eca-109 cells in nude mice. |
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