Tet-on系统调控A20基因在鼻咽癌肝转移亚系中的表达

Regulating A20 Gene Expression in Hepatic Metastic Subline of Nasopharyngeal Carcinoma by Tet-on System

目的构建pSUPERIOR-A20可调控性载体,检测其在肝转移亚系中表达的可调控性。方法通过基因芯片筛选出鼻咽癌肝转移相关基因,结合文献调研获得头颈部癌转移标签基因,并通过荧光定量PCR检测发现A20可能在鼻咽癌肝转移过程中发挥了重要作用。在线设计A20特异性干扰片段,选择相应酶切位点后将其插入pSUPERIOR质粒中,应用酶切鉴定和序列分析方法验证所构建载体的正确性。将pSUPERIOR质粒和Tet-on质粒共转染肝转移亚系5-8F-H3,PCR检测A20的表达。结果Real-time PCR检测A20在肝转移亚系5-8F-H3中的表达显著上调,结果具有统计学意义(P=0.005)。且成功构建了pSUPERIOR-A20载体,与Tet-on质粒共转染5-8F-H3后经嘌呤霉素筛选克隆,经荧光定量PCR检测阳性克隆A20基因的表达显著下调。结论成功构建pSUPERIOR-A20载体,并可有效干扰鼻咽癌肝转移亚系5-8F-H3中A20基因的表达,为进一步研究A20在鼻咽癌肝转移中的作用奠定了良好的基础。

Objective To construct inducible vector pSUPERIOR-A20 and study the expression character of A20 under the regulation of Dox in Hepatic Metastic Subtine of Nasopharyngeal Carcinoma （NPC）. Method The liver metastasis-related genes in NPC were screened by DNA microarray and metastasis signature genes of head and neck cancer. A20 were validated with significant higher expression by Quantitative RT-PCR. Two different siRNA targeting against human A20 gene were designed and then inserted into pSUPERIOR. The recombinant plasmids pSUPERIOR- A20 was confirmed by restriction endonuclease analysis and DNA sequencing. 5-8F-H3 cells were co-transfected with Tet-on and pSUPERIOR-A20 and the expression of A20 was detected by PCR. Results A20 was validated with significant higher expression in 5-SF-H3 by Quantitative RT-PCR. pSUPERIOR-A20 vector was constructed successfully and co-transfected into 5-8F-H3 with Tet-on. The positive clones were screened by Puromycin. The down regulation of A20 was confirmed by Quantitative RT-PCR. Conclusion The successful construction of inducible vector pSUPERIOR-A20 can inhibit A20 expression in 5-8F-H3, which may provide the fine basis for further study of liver metastasis of NPC.