TNF-α激活Wnt信号通路促进人结肠癌干细胞侵袭

TNF-α activates Wnt signaling pathway to promote the invasion of human colon cancer stem cells

目的探讨炎症环境下Wnt信号通路对人结肠癌干细胞侵袭的影响。方法以人结肠癌细胞系HT29细胞为材料,用流式细胞荧光分选技术(FACS)分选CD44+CD133+的人结肠癌干细胞;流式细胞术及单细胞克隆形成实验对分选出的干细胞进行鉴定;肿瘤坏死因子α(TNF-α)处理CD44+CD133+HT29细胞建立炎症细胞模型,噻唑蓝(MTT)实验筛选TNF-α作用的最适剂量及最佳作用时间; Wnt信号通路抑制剂Dickkopf相关蛋白1(DKK1)作用于CD44+CD133+HT29炎症细胞,实验分为空白组、TNF-α处理组、加DKK1的TNF-α处理组。MTT实验检测各组细胞增殖情况,Western blot法检测Wnt信号通路中相关蛋白β联蛋白(β-catenin)、细胞周期蛋白D1(cyclin D1)、c-Myc及上皮间质转化(EMT)相关蛋白上皮钙黏素(E-cadherin)、波形蛋白(vimentin)的表达情况,TranswellTM侵袭实验检测各组细胞侵袭情况。结果流式细胞分选术成功分选出结肠癌CD44+CD133+HT29干细胞。MTT实验筛选出TNF-α作用的最适剂量为10 ng/m L,最佳作用时间为48 h。与空白组相比,TNF-α处理组中CD44+CD133+HT29细胞的相对存活率增加,TranswellTM穿膜的细胞数增加,β-catenin、cyclin D1、c-Myc表达升高,E-cadherin表达降低、vimentin表达升高;与TNF-α处理组相比,加DKK1的TNF-α处理组CD44+CD133+HT29细胞的相对存活率降低,TranswellTM穿膜细胞数减少,β-catenin、cyclin D1、c-Myc表达降低,E-cadherin表达升高、vimentin表达降低。结论TNF-α通过激活Wnt信号通路促进结肠癌干细胞的侵袭能力。

Objective To explore the effect of Wnt/β-catenin signal pathway on the invasion of human colon cancer stem cells in the inflammatory environment. Methods The CD44^+CD133^+human colon cancer stem cells were sorted from HT29 human colon cancer cell line by fluorescence-activating cell sorting( FACS),and these stem cells were identified by flowcytometry and single cell cloning formation assay. The inflammatory cell model was established by tumor necrosis factor-α( TNF-α) treating CD44^+CD133^+HT29 cells and the optimal dose and reaction time of TNF-α were confirmed by MTT assay.DKK1 as an inhibitor of the Wnt signaling pathway was used in the inflammatory cell model. The experiment cells weredivided into control group,TNF-α treatment group and TNF-α combined with DKK1 group. The cel proliferation was determinedby MTT assay,and the expression of Wnt signaling pathway-related proteins including β-catenin,cyclin D1,c-Myc and epithelialmesenchymal transition( EMT)-related proteins( E-cadherin,vimentin) were detected by Western blot analysis. Theinvasive ability of cancer stem cells was detected by TranswellTMassay. Results The CD44^+CD133^+human colon cancerstem cells were successfully obtained from HT29 cells. Compared with the control group,the relative survival rate andinvasive ability of CD44^+CD133^+HT29 cells increased after treated with 10 ng/m L TNF-α for 48 hours,and the expression ofE-cadherin was downregulated and vimentin was upregulated after CD44^+CD133^+HT29 cells were treated with TNF-α.Compared with the TNF-α treatment group,the relative survival rate of CD44^+CD133^+HT29 cells was reduced after DKK1 treatment. The number of transmembrane cells and the expression of Wnt signaling pathway-related proteins includingβ-catenin,cyclin D1 and c-Myc decreased after DKK1 treatment,and E-cadherin expression was upregulated and vimentinexpression was reduced after CD44^+CD133^+HT29 cells were treated with DKK1. Conclusion TNF-α promotes the invasionof colon cancer stem cells by activating the Wnt signaling pathway.