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目的预测并初步鉴定转录因子对hsa-miR-206启动子区的靶向结合,以进一步研究其转录调控机制。方法通过生物信息学软件预测Myo D在hsa-miR-206启动子区有结合位点,分别构建p GL3-miR206 promoter萤光素酶报告载体和p EGFP-N2-Myo D表达质粒,分组共转染于He La细胞,应用双萤光素酶报告基因法检测定萤光素酶活性。结果生物信息学软件预测hsa-miR-206启动子区有2个转录因子Myo D结合位点。构建的2个载体经电泳及测序证实无误。双萤光素酶报告基因法检测显示,与对照组相比,共转染p GL3-Basic-miR206 promoter荧光素酶报告载体和p EGFP-N2-Myo D表达质粒实验组的荧光素酶活性显著降低,差异有统计学意义(P<0.05)。结论 Myo D能在hsa-miR-206启动子区靶向结合,是为hsa-miR-206上游转录因子,为后续研究miR-206的转录调控机制提供了一定的实验基础。
OBJECTIVE: To predict and preliminarily identify the target binding of transcription factor to hsa-miR-206 promoter region to further study its transcriptional regulation mechanism. Methods Bioinformatics software was used to predict the binding site of Myo D in the promoter region of hsa-miR-206. The pGL3-miR206 promoter luciferase reporter plasmid and the p EGFP-N2-Myo D expression plasmid were constructed respectively, Staining in HeLa cells, luciferase activity was measured using a dual luciferase reporter assay. Results The bioinformatics software predicted two Myo D binding sites in the hsa-miR-206 promoter region. The constructed 2 vectors were confirmed by electrophoresis and sequencing. Luciferase reporter assay showed that compared with the control group, the luciferase activity of the co-transfected pGL3-Basic-miR206 promoter luciferase reporter vector and the p EGFP-N2-Myo D expression plasmid experimental group was significantly Reduce, the difference was statistically significant (P <0.05). Conclusion Myo D can target-bind in the promoter region of hsa-miR-206, which is the upstream transcription factor of hsa-miR-206, providing experimental basis for further study on the transcriptional regulation of miR-206.