体轴抑制因子1敲除及过表达对ACT-1人未分化甲状腺癌细胞中B细胞淋巴瘤-2表达影响的初步研究

Preliminary Study on the Effect of Axis Inhibitor 1 Knockout and Overexpression on the Expression of B Cell Lymphoma-2 in ACT-1 Human Anaplastic Thyroid Cancer Cells

目的探讨体轴抑制因子1(AXIN1)的敲除及过表达对ACT-1人未分化甲状腺癌细胞中B细胞淋巴瘤-2(Bcl-2)影响及可能机制。方法采用实时荧光定量PCR(RT-qPCR)法检测ACT-1人未分化甲状腺癌细胞与Nthy-ori-3-1人正常甲状腺细胞中AXIN1 mRNA水平表达差异。采用体外分子克隆技术及过表达技术构建AXIN1基因过表达ACT-1细胞系,RT-qPCR及蛋白质免疫印迹法(WB)分别检测其AXIN1 mRNA及蛋白表达水平。在前期构建成功的AXIN1敲除的ACT-1单克隆细胞基础上,结合本实验构建的过表达细胞系,采用RT-qPCR及WB分别检测两组细胞中Bcl-2 mRNA和蛋白表达水平。结果与对照组相比,ACT-1中AXIN1表达降低,过表达组AXIN1 mRNA和蛋白表达水平增加,过表达组Bcl-2 mRNA和蛋白表达水平降低,而敲除组Bcl-2 mRNA和蛋白表达水平均增高(P<0.05)。结论敲除及过表达AXIN1基因可能通过改变Bcl-2的表达来影响ATC肿瘤发生过程。

Objective To investigate the effect of knockout and overexpression of axis inhibitor 1(AXIN1)on B-cell lymphoma-2(Bcl-2)in ACT-1 human anaplastic thyroid cancer cells and its possible mechanism.Methods Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the difference in AXIN1 mRNA expression between ACT-1 cells and Nthy-ori-3-1 cells;molecular cloning technologyin vitro and overexpression technology were used to construct the AXIN1 overexpressing ACT-1 cell line,the AXIN1 mRNA and protein expression levels were detected by RT-qPCR and Western blotting(WB)respectively;based on the successful AXIN1 knockout ACT-1 monoclonal cells constructed in the previous experiments,combined with the overexpression cell line constructed in this experiment,RT-qPCR and WB were used to detect the expression levels of Bcl-2 mRNA and protein between the two groups.Results Compared with the control group,the expression of AXIN1 in ACT-1 was reduced,the mRNA and protein expression levels of AXIN1 in the overexpression group were increased,the expression levels of Bcl-2 mRNA and protein in the overexpression group were decreased,and the Bcl-2 mRNA and protein expression levels in the knockout group were increased(P<0.05).Conclusion Knockout and overexpression of AXIN1 gene may affect the process of ATC tumorigenesis by changing the expression of Bcl-2.