TNF-α通过CXCL10/CXCR3信号通路促进结肠癌细胞上皮间质化的相关分子机制

Molecule mechanisms of TNF⁃αpromoting the epithelial mesenchymal transformation of colon cancer cells through the CXCL10/CXCR3 signaling pathway

目的 探讨TNF-α通过调控结肠癌SW480细胞CXCL10/CXCR3轴的表达影响上皮间质化(EMT)及其分子机制。方法 培养人结肠癌SW480细胞,分别通过TNF-α、siRNA-CXCR3处理细胞,将细胞分为对照组(NC组)、TNF-α刺激组(TNF-α组)及TNF-α刺激+siRNA-CXCR3沉默组(TNF-α+si-CXCR3组)。通过Real-time PCR检测各组细胞CXCL10、CXCR3及上皮间质化相关基因的mRNA表达量;Western Blot检测各组细胞CXCL10/CXCR3通路蛋白及上皮间质化相关蛋白表达量的影响;免疫组化检测细胞中上EMT上皮标志物上皮细胞钙粘蛋白(E-cad)与EMT间质标志物波形蛋白(Vimentin)的变化情况;划痕实验检测各组细胞的迁移能力;Transwell实验检测各组细胞的侵袭能力。结果 TNF-ɑ组细胞中通路基因CXCL10、CXCR3的m RNA水平高于NC组,差异有统计学意义(P<0.05),TNF-α+si-CXCR3组细胞中CXCR3水平低于TNF-α组,差异有统计学意义(P<0.05);TNF-ɑ组细胞中CXCL10、CXCR3、Vimentin与Fibronectin的蛋白表达量高于NC组,E-cad的蛋白表达量低于NC组,差异有统计学意义;TNF-α+si-CXCR3组细胞CXCR3、Vimentin与Fibronectin的蛋白表达量低于TNF-ɑ组,E-cad的蛋白水平高于TNF-ɑ组;TNF-ɑ组中Vimentin的表达量高于NC组,E-cad的表达量低于NC组,差异有统计学意义(P<0.05),TNF-α+siCXCR3组中Vimentin的表达量低于TNF-ɑ组,差异有统计学意义(P<0.05),E-cad的表达量高于TNF-ɑ组;TNF-ɑ组迁移能力高于NC组,差异有统计学意义(P<0.05),TNF-α+si-CXCR3组细胞迁移能力低于TNF-ɑ组,差异有统计学意义(P<0.05)。TNF-ɑ组细胞的侵袭能力高于NC组,差异有统计学意义(P<0.05),TNF-α+si-CXCR3组细胞侵袭能力低于TNF-ɑ组,差异有统计学意义(P<0.05)。结论 TNF-α能够诱导结肠癌SW480细胞发生上皮间质化,并促进其迁移、侵袭水平,这一过程可能与激活CXCL10/CXCR3信号通路有关。

Objective To investigate the impact of TNF-αon epithelial mesenchymal transforma-tion(EMT)and its molecular mechanism by regulating the expression of CXCL10/CXCR3 axis in colon can-cer SW480 cells.Methods SW480 cells were cultured and treated with TNF-αand siRNA-CXCR3,respec-tively.The mRNA expression levels of CXCL10 and CXCR3 were detected using real-time PCR.Western Blot was performed to detect the expression levels of proteins involved in the CXCL10/CXCR3 pathway and EMT.Immunohistochemistry was used to observe changes in the expression of the EMT epithelial marker E-cadherin and the EMT interstitial marker vimentin.Transwell assay was conducted tp assess the migration and invasion abilities of cells in each group.Results The mRNA levels of CXCL10 and CXCR3 in the NF-α+si-CXCR3 group were higher than those in the NC group,and the difference was statistically significant(P<0.05).Addi-tionally,the CXCR3 levels in the TNF-α+si-CXCR3 group were lower than those in the TNF-αgroup,and this difference was also statistically significant(P<0.05).The protein expression levels of CXCL10,CXCR3,vimen-tin and Fibronectin in the TNF-ɑgroup were higher than those in the NC group.However,the protein expression levels of E-CAD were lower than those in the NC group,and the difference was statistically significant(P<0.05).In the NF-α+si-CXCR3 group.The protein expression levels of CXCR3,vimentin and Fibronectin were lower than those in the TNF-ɑgroup.The protein level of E-CAD was higher than that in the TNF-ɑgroup.The expres-sion of vimentin in the TNF-ɑgroup was higher than that of the NC group,and the expression of E-cad was lower than that of the NC group.The differences were statistically significant(P<0.05).The expression of vimen-tin in the TNF-α+si-CXCR3 group was lower than that in the TNF-ɑgroup,and the difference was statistically sig-nificant(P<0.05).The expression of E-CAD was higher than that of the TNF-ɑgroup.The migration ability of the TNF-ɑgroup was higher than that of the NC group,and the difference was statistically significant(P<0.05).The cell migration ability of the TNF-α+si-CXCR3 group was lower than that of the TNF-ɑgroup,and the difference was statistically significant(P<0.05).The invasive ability of cells in the TNF-ɑgroup was higher than that of the NC group,and the difference was statistically significant(P<0.05).The cell invasion ability of the TNF-α+si-CXCR3 group was lower than that of the TNF-ɑgroup,and the difference was statistically significant(P<0.05).Conclusion TNF-αcould induce epithelialization of colon cancer SW480 cells and promote their migration and invasion levels.This effect may be related to the activation of the CXCL10/CXCR3 signaling pathway.