小鼠pre-miRNA-122启动子荧光素酶报告质粒的构建及表达调控

Construction and expression regulation of mouse pre-miRNA-122 gene promoter luciferase reporter plasmids

[目的]构建小鼠pre-miRNA-122基因启动子荧光素酶报告质粒,研究血小板衍生生长因子(Platelet-derived growth factor,PDGF)对miRNA-122表达的影响。[方法]利用生物信息学分析pre-miRNA-122启动子片段并扩增,插入pGL3-basic载体中获得报告质粒pmir-122-luc。将pmir-122-luc转染小鼠Huh-7和HepG2细胞,24h后检测荧光素酶活性;同样将其转染小鼠肝星状细胞(Hepatic Stellate Cells,HSCs),PDGF处理12h后检测活性。[结果]pre-miRNA-122上游5.5~4.5 kb为启动子区域。构建的质粒经酶切、测序鉴定正确。pmir-122-luc荧光素酶活性较pGL3-basic显著增加,且在Huh-7细胞高表达(24±5.02倍)而在HepG2细胞低表达(1.8±0.34倍)。pmir-122-luc能在HSCs中表达,且PDGF处理后活性降低(P<0.05)。[结论]小鼠pre-miRNA-122启动子荧光素酶活性报告质粒构建成功;PDGF抑制miRNA-122在HSCs中表达,为探索miRNA-122的表达调控机制提供了新的窗口。

[ Objective] To construct mouse pre -miRNA -122 gene promoter luciferase reporter plasmids and study effect of Platelet- derived growth factor （PDGF） on the miRNA - 122 expression. [ Methods] Pre -miRNA -122 gene promoter se- quence was analyzed by bioinformatics and amplified by PCR. Product of PCR was inserted into pGL3 -basic vector,and a luciferase reporter plasmid, pmir - 122 -luc, was obtained. The constructed plasmids were transiently transfected into mouse liver cancer cell lines HepG2 and Huh 7, and the luciferase activity was determined 24 h after transfection. Mouse Hepatic Stellate Cells （HSCs） transfected with plasmids in the same way, and were collected 12h later for determination of luciferase activity af- ter PDGF treatment. [ Results ] The promoter was located in the region of 5.5 - 4.5 kb on the 5 ＇ - untranslated region of mouse pre - miRNA - 122 gene. Both restriction analysis and sequencing proved that plasmid pmir - 122 - lue was constructed correctly. The luciferase activity of pmir - 122 - luc was significantly increased comparing to pGL3 - basic vector, and in Huh - 7 cells with high expression （24 ±5.02 times） and low expression in HepG2 cells （ 1.8 ±0.34 times）, pmir - 122 - luc could be expressed in HSCs, and luciferase activity was obviously decreased by PDGF （P 〈0.05 ）. [ Conclusion] Mouse pmir -122 -luc was constructed successfully;PDGF plays a key role in inhibition of miRNA- 122 expression in HSCs.