摘要
目的研究发现,HMGA2促进肿瘤细胞发生EMT与其下游基因SOX7有关。为进一步验证转录因子HMGA2与其下游基因SOX7之间的调控关系,本研究通过构建人SOX7基因启动子荧光素酶报告质粒,采用荧光素酶报告实验观察HMGA2对SOX7基因启动子荧光素酶活性的影响,为研究HMGA2转录表达的调控机制提供必要的实验基础。方法采用PCR技术扩增人SOX7基因启动子序列(-1 549^+79nt),将SOX7基因启动子插入荧光素酶报告基因载体pGL3-basic中构建质粒pGL3-SOX7-promoter;再分别将pGL3-SOX7-promoter、pGL3-basic-SOX7-promoter和对照质粒(pLV-UbC-IRES2-EGFP)、pGL3-SOX7-promoter和HMGA2表达质粒(pLV-UbC-HMGA2-IRES2-EGFP)3组质粒共转染293T细胞,培养48h后,检测3组荧光素酶的表达情况,观察HMGA2对SOX7基因启动子的调控作用。结果通过菌落PCR和核酸测序证实,人SOX7基因启动子荧光素酶报告质粒pGL3-SOX7-promoter构建成功;将pGL3-SOX7-promoter和HMGA2表达质粒(pLV-UbC-HMGA2-IRES2-EGFP)共转染293T细胞48h后,与对照组相比,SOX7基因启动子介导荧光素酶的活性降低约31%,受到明显抑制(P=0.003),提示HMGA2可能调控SOX7基因启动子的活性。结论本研究成功构建了SOX7基因启动子荧光素酶报告质粒,初步验证了SOX7是HMGA2下游的作用靶点。
OBJECTIVE It was found that HMGA2 might be relevant to its downstream gene SOX7 in the EMT process of turnout cells. The objective of this study was to further verify the relationship between transcription factor HMGA2 and its downstream gene, by constructing the human SOX7 promoter luciferase report plasmid and the luciferase reporter experiment, to observe the effects of HMGA2 on SOX7 promoter luciferase activity and thus provide the necessary experimental basis for researching on the transcriptional and regulation mechanism of HMGA2. METHODS The hu-man SOX7 gene promoter sequences （-1 549 - ＋79nt） were amplified by PCR, and SOX7 gene promoter was inserted into the luciferase reporter gene vector pGL3 basic. Three group plasmids, SOX7 promoter luciferase reporter plasmid （pGL3-SOX7-promoter）, pGL3-SOX7 promoter and the control plasmid （pLV-UbC-IRES2-EGFP）, pGL3 SOX7-pro-moter and HMGA2 expression plasmid （pLV-UbC-HMGA2-IRES2-EGFP）, were respectively co-transfected into 293T cells, and then we measured the expression of luciferase after 48 hours and observed the promoting effect of HMGA2 on SOX7. RESULTS It was verified that human SOX7 gene promoter luciferase reporter plasmid was successfully constructed by PCR and nucleotide sequencing. Compared with the control group, SOX7 promoter activity was significantly suppressed about 31% after pC-L3-SOX7-promoter and pLV UbC-HMGA2-IRES2-EGFP were co-transfected into 293T cells （P〈0.01）, which indicated that HMGA2 may regulate SOX7 promoter activity. CONCLUSION SOX7 gene pro moter luciferase reporter plasmid was constructed successfully in this study, and it is preliminarily verified that S（）X7 gene may he the downstream target of HMGA2.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2016年第19期1282-1286,共5页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(81272753)
