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目的检测siRNA干扰Oct4基因对P19细胞中miR-302启动子活性的影响,探讨维持胚胎癌(EC)细胞及胚胎干细胞(ES)全能性的调控机制。方法采用Western blotting检测Oct4 siRNA对Oct4蛋白的干扰效果;采用双荧光素酶报告基因分析系统检测siRNA干扰Oct4基因对P19细胞中miR-302基因启动子活性的影响。结果对于Oct4蛋白的最适干扰量为2.0μg siRNA1和2.0μg siRNA2;向P19细胞中转染Oct4 siRNA可以降低miR-302启动子的荧光素酶活性。结论 Oct4对miR-302的启动子具有正向调控作用。
Objective To detect the effect of siRNA interfering Oct4 gene on the activity of miR-302 promoter in P19 cells and to explore the regulatory mechanism of maintaining the pluripotency of embryonic carcinoma (EC) cells and embryonic stem cells (ES). Methods The effect of Oct4 siRNA on Oct4 protein was detected by Western blotting. The effect of Oct4 siRNA on the promoter activity of miR-302 gene in P19 cells was detected by dual luciferase reporter gene assay system. The results showed that the optimal amount of interference for Oct4 protein was 2.0μg siRNA1 and 2.0μg siRNA2. Transfection of Oct4 siRNA into P19 cells could reduce the luciferase activity of miR-302 promoter. Conclusion Oct4 has a positive regulatory effect on miR-302 promoter.