胚胎癌F9细胞中Oct4对miR-302启动子的调控作用

Oct4 regulates the miR-302 promoter in F9 mouse embryonic carcinoma cells

目的:检测胚胎癌F9细胞中Oct4对miR-302启动子活性是否具有调节作用。方法:构建PGL3-P-miR-302启动子荧光素酶报告基因质粒并验证其活性,然后将0.5μg PGL3-P-miR-302分别与0.5和1.0 g Oct4表达质粒共转染至293T细胞中,检测过表达Oct4对miR-302启动子活性的影响;并将0.5μg PGL3-P-miR-302和Oct4 siRNA(2.0μg siRNA1和2.0μg siRNA2)共转染到F9细胞中,检测Oct4 RNA干扰对miR-302启动子活性的影响,以验证Oct4对miR-302启动子是否有调控作用。结果:成功构建了PGL3-P-miR-302启动子荧光素酶报告基因质粒,并证实了PGL3-P-miR-302在F9细胞中具有启动子活性;在T293细胞中,当Oct4表达质粒为0.5μg时miR-302启动子转录活性是对照组的2.6倍(P<0.05),当Oct4为1.0μg时miR-302启动子转录活性是对照组的4.3倍(P<0.05);在F9细胞中miR-302启动子的活性在转染Oct4 siRNA后明显下降,约为siRNA非特异性对照组的68%(P<0.05)。结论:Oct4对miR-302启动子具有正调节作用,miR-302可能是Oct4的靶基因之一。

To investigate whether Oct4 could activate miR-302 promoter in the F9 mouse embryonic carcinoma cells. METHODS：PGL3-P-miR-302 was created by cloning the PCR-amplified region of the miR-302 putative promoter. PGL3-P-miR-302 promoter reporter plasmids （0.5 μg） were co-transfected with 0.5μg or 1.0μg Oct4 expression vector into 293T cells. PGL3-P-miR-302 promoter reporter plasmids （0.5μg） were co-transfected with Oct4 siRNA（2.0μg siRNA1 and 2.0μg siRNA2） into F9 cells. Luciferase activity was assayed. RESULTS：PGL3-P-miR-302 was created,and the cell-specific activity of this promoter was identified by its transfection into F9 cells. With 0.5 μg and 1.0 μg Oct4 overexpression in 293T cells,miR-302 promoter activity increased 2.6-fold and 4.3-fold,respectively. After siRNA-mediated knockdown of Oct4,miR-302 luciferase activity fell to 68% compared with control siRNA. CONCLUSION：This study showed that the miR-302 cluster acted downstream of the Oct4 regulation network in F9 cells.