Cloning and characterization of the 5' flanking region of microRNA let-7a-1/let-7f-1 gene cluster in human lung cancer cell

Abstract

In order to elucidate the molecular basis of microRNA let-7a-1/let-7f-1 gene cluster, the transcription initiation site which was determined by 5’ rapid amplification of cDNA ends (5’RACE) and 2.1 kb of the 5' flanking region proximal to the pre-let-7a-1 was isolated and characterized. The promoter activity of the 2.1 kb fragment was analyzed by a firefly luciferase-encoding gene expression vector (pGL3) transiently transfected into lung cancer cell line A549. The 2.1 kb promoter of let-7a-1/let-7f-1 displayed a lower activity and was significantly enhanced by ectopic expression of c/EBPα or p53 and treatment with dexamethasone. Despite the induction of other let-7 family members such as let-7a-3, let-7c and let-7d, all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA) display little enhancement effect on 2.1 kb promoter of let-7a-1/let-7f-1, as well as 1,25-(OH)₂D₃.

Key words: let-7a-1; let-7f-1, 5’ rapid amplification of cDNA ends (5’RACE), promoter, lung cancer.