敲减ATF3对结直肠癌肿瘤干细胞自我更新与上皮间质转化及免疫抑制因子分泌的影响

Knockdown of ATF3 affects the self-renewal and epithelial-mesenchymal transition of colorectal cancer stem cells and the secretion of immunosuppressive molecules

目的研究敲减活化转录因子3(ATF3)对结直肠癌肿瘤干细胞(CRC-CSCs)自我更新能力与上皮间质转化的影响,并检测免疫抑制因子分泌的变化。方法培养人结直肠癌细胞系HCT116,通过流式细胞术分选CD133+表达的CRC-CSCs细胞,转染sh-ATF3慢病毒载体至CRC-CSCs细胞作为实验组(sh-ATF3组),以转染sh-NC慢病毒载体作为阴性对照组(sh-NC组),正常培养的CRC-CSCs细胞作为对照组。实时荧光定量PCR(qRT-PCR)检测ATF3相对表达量,CCK-8法检测细胞活性变化,成球实验检测细胞生长更新能力,平板克隆形成实验检测细胞克隆形成能力,免疫荧光染色和蛋白质免疫印迹(Western Blot)法检测E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达,酶联免疫吸附法(ELISA法)检测细胞分泌免疫抑制因子TGF-β1、VEGF、IL-6、IL-10的含量变化,Western Blot检测自杀相关因子(Fas)、Fas配体(FasL)蛋白表达水平。结果对照组比较,sh-ATF3组CRC-CSCs细胞中ATF3 mRNA相对表达量下降(P<0.05),转染后48 h和72 h细胞活性降低(P<0.05),细胞成球能力与克隆形成能力均下降(P<0.01),细胞中E-cadherin蛋白表达较高而Vimentin蛋白表达较低(P<0.05),同时,细胞上清液中TGF-β1、VEGF、IL-10的含量均明显下降(P<0.01),Fas蛋白表达升高,FasL蛋白表达下降(P<0.05)。结论敲减ATF3可抑制结直肠癌肿瘤干细胞自我更新与上皮间质转化,下调免疫抑制因子TGF-β1、VEGF、IL-10表达,并且上调Fas蛋白,下调FasL蛋白的表达。

Objective To study the effect of knockdown activating transcription factor 3(ATF3)on the self-renewal ability and epithelial-mesenchymal transition of colorectal cancer tumor stem cells(CRC-CSCs),and detect the changes in the secretion of immunosuppressive factors.Methods Culture the human colorectal cancer cell line HCT116,Sort CD133+expressing CRC-CSCs cells by flow cytometry,transfect sh-ATF3 lentiviral vector to CRC-CSCs cells as the experimental group(sh-ATF3),use sh-NC lentiviral vector as a negative control group(sh-NC),normally cultured CRC-CSCs cells served as a control group,real-time fluorescent quantitative PCR(qRT-PCR)detects the relative expression of ATF3,CCK-8 method detects changes in cell activity,spheroidization test detects cell growth and renewal ability,plate clone formation test detects cell clone formation ability,immunofluorescence staining and Western Blot method were used to detect the expression of E-cadherin and Vimentin,enzyme-linked immunosorbent assay(ELISA method)detects changes in the levels of immunosuppressive factors TGF-β1,VEGF,IL-6,and IL-10 secreted by cells,western Blot detects the protein expression levels of suicide-related factors(Fas)and Fas ligand(FasL).Results Compared with the control group,the relative expression of ATF3 mRNA in CRC-CSCs cells in the sh-ATF3 group decreased(P<0.05),and cell viability decreased at 48 h and 72 h after transfection(P<0.05),the spheroidizing ability and the cloning ability of the cells decreased(P<0.01),the expression of E-cadherin protein in the cells was higher and the expression of Vimentin protein was lower(P<0.05),at the same time,the contents of TGF-β1,VEGF,and IL-10 in the cell supernatant were significantly decreased(P<0.01),Fas protein expression increased,and FasL protein expression decreased(P<0.05).Conclusion Knockdown of ATF3 can inhibit colorectal cancer stem cell self-renewal and epithelial-mesenchymal transition,down-regulate the expression of immunosuppressive factors TGF-β1,VEGF,IL-10,and up-regulate the expression of Fas protein and down-regulate the expression of FasL protein.