CAJ | 학술논문

目的 miRNA- 122启动子序列的预测、克隆及特异性分析.方法分别从肝癌细胞系Huh-7及HepG2中扩增出预测的miR-122启动子,并将其克隆至含荧光素酶(firefly luciferase,Fluc)报告基因pGL4.17质粒上,用重组质粒转染Huh-7、HepG2及HeLa细胞,分析启动子的特性.结果成功预测并克隆出miR-122的启动子序列.重组质粒转染细胞后,启动子能够启动Fluc的表达.用含启动子P1的重组载体pGL4.17-P1转染HeLa细胞后,用两种荧光素酶检测体系测得重组载体中荧光素酶的表达水平显著高于对照组(t =0.000 21,P＜0.01及t=0.000 38,P＜0.01).结论 Huh-7、HepG2细胞中miR-122表达差异与启动子序列缺失无关.而受miR-122启动子调节的报告基因在非肝细胞系(HeLa)中也表达,表明miR-122启动子不具有肝细胞特异性.
목적 miRNA- 122계동자서렬적예측、극륭급특이성분석.방법 분별종간암세포계Huh-7급HepG2중확증출예측적miR-122계동자,병장기극륭지함형광소매(firefly luciferase,Fluc)보고기인pGL4.17질립상,용중조질립전염Huh-7、HepG2급HeLa세포,분석계동자적특성.결과 성공예측병극륭출miR-122적계동자서렬.중조질립전염세포후,계동자능구계동Fluc적표체.용함계동자P1적중조재체pGL4.17-P1전염HeLa세포후,용량충형광소매검측체계측득중조재체중형광소매적표체수평현저고우대조조(t =0.000 21,P＜0.01급t=0.000 38,P＜0.01).결론 Huh-7、HepG2세포중miR-122표체차이여계동자서렬결실무관.이수miR-122계동자조절적보고기인재비간세포계(HeLa)중야표체,표명miR-122계동자불구유간세포특이성.
Objective To predict and clone miR-122,and to analyze its specificity.Methods The putative promoter regions of miR-122 were extracted from hepatocarcinoma cell lines,Huh-7 and HepG2,and cloned into plasmid pGL4.17,which contains the reporter gene of Firefly luciferase (Fluc).The transcription-promoting capacity of these putative promoters of miR-122 was detected.Results We successfully predicted and cloned miR-122 promoter sequences.The transcription-promoting capacity of the putative promoter of miR-122 was detected after it was transfected into the cells.The expression of Fluc in HeLa cells transfected with the constructed plasmid pGL4.17-P 1,which contains the putative promoter 1 of miR-122 ( P1 ),was significantly increased than that in the control cells ( t =0.000 21,P ＜ 0.01 ; t =0.000 38,P ＜ 0.01 ) detected by Dual and Single-Luciferase reporter assay system,respectively.Conclusion The distinction of miR-122 expression in cell lines of Huh-7 and HepG2 was not related with the presence of miR-122 promoters.Reporter gene controlled by the promoter of miR-122 was also expressed in non-hepatic cells.These results suggested that the promoters of miR122 showed no hepatic specificity.