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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。
The purpose of the study found that HMGA2 promote tumor cell EMT and its downstream gene SOX7. In order to further verify the regulation of the transcription factor HMGA2 and its downstream gene SOX7, luciferase reporter assay was used to detect the effect of HMGA2 on the luciferase activity of SOX7 promoter by constructing luciferase reporter plasmid of human SOX7 promoter. , Providing the necessary experimental basis for studying the regulation mechanism of HMGA2 transcriptional expression. Methods The promoter sequence of human SOX7 gene (-1 549 ~ + 79 nt) was amplified by PCR. The promoter of SOX7 gene was inserted into the luciferase reporter gene vector pGL3-basic to construct the plasmid pGL3-SOX7-promoter. The plasmids of pGL3-SOX7-promoter, pGL3-basic-SOX7-promoter and control plasmid pLV-UbC-IRES2-EGFP, pGL3-SOX7-promoter and HMGA2 expression plasmid (pLV-UbC-HMGA2-IRES2-EGFP) 293T cells were cultured 48h after the detection of three groups of luciferase expression observed HMGA2 SOX7 gene promoter regulatory effect. Results The colony PCR and nucleic acid sequencing confirmed that luciferase reporter plasmid pGL3-SOX7-promoter of human SOX7 promoter was successfully constructed. The pGL3-SOX7-promoter and HMGA2 expression plasmid (pLV-UbC-HMGA2-IRES2- EGFP) Compared with the control group, SOX7 promoter-mediated luciferase activity was reduced by about 31% (P = 0.003), indicating that HMGA2 may regulate the activity of SOX7 gene promoter after 48h incubation of 293T cells. Conclusion This study successfully constructed the SOX7 gene promoter luciferase reporter plasmid, preliminary verified that SOX7 is the downstream target of HMGA2.