CAJ | 학술논문

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。
목적：검측배태암F9세포중Oct4대miR-302계동자활성시부구유조절작용。방법：구건PGL3-P-miR-302계동자형광소매보고기인질립병험증기활성，연후장0.5μg PGL3-P-miR-302분별여0.5화1.0 g Oct4표체질립공전염지293T세포중，검측과표체Oct4대miR-302계동자활성적영향；병장0.5μg PGL3-P-miR-302화Oct4 siRNA(2.0μg siRNA1화2.0μg siRNA2)공전염도F9세포중，검측Oct4 RNA간우대miR-302계동자활성적영향，이험증Oct4대miR-302계동자시부유조공작용。결과：성공구건료PGL3-P-miR-302계동자형광소매보고기인질립，병증실료PGL3-P-miR-302재F9세포중구유계동자활성；재T293세포중，당Oct4표체질립위0.5μg시miR-302계동자전록활성시대조조적2.6배(P<0.05)，당Oct4위1.0μg시miR-302계동자전록활성시대조조적4.3배(P<0.05)；재F9세포중miR-302계동자적활성재전염Oct4 siRNA후명현하강，약위siRNA비특이성대조조적68%(P<0.05)。결론：Oct4대miR-302계동자구유정조절작용，miR-302가능시Oct4적파기인지일。
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.