title: Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines.
abstract: Recent studies have revealed that microRNAs have a strong association with cancer in humans. The miRNA let-7 is highly expressed in normal lung tissue, but frequently expressed at reduced levels in lung cancers. Let-7a2 is a member of the let-7 family. So far, little is known about the transcriptional regulation of let-7a2. Our study is focused on the characterization and functional analysis of the promoter of the human miRNA let-7a2 in A549 cell lines. Firstly, 5' rapid amplification of cDNA ends (5' RACE) was carried out and a 2.8 kb fragment in the upstream of let-7a2 gene was then cloned into pGL3-basic vector. Sequence analysis with the MatInspector database revealed that there were putative binding sites for some important transcriptional factors in the promoter region of let-7a2, such as p53, c-Myc, Ras, CEBPα, RORA, RXR, TCF, and GR. Additionally, a series of transfection and luciferase reporter assays were carried out to test let-7a2 promoter activity. RT-PCR and transfection of let-7a target sequence-reporter plasmid were performed to detect transcription levels of the let-7a2 gene in A549 cells treated with 9-cis-RA, all-trans-RA, lithium chloride or dexamethasone. Our results showed that the recombinant pGL3-p7a2 could acts as a promoter. The promoter activity of the 2.8 kb fragment could be downregulated by transfection with CEBPα or treatment with lithium chloride and enhanced by 9-cis-RA or all-trans-RA treatment. Furthermore, the results of RT-PCR analysis and transfection of let-7a target sequence-reporter plasmid showed that 9-cis-RA and all-trans-RA both upregulated let-7a2 expression, while lithium chloride downregulated its expression. Our results suggest that 9-cis-RA, all-trans-RA,lithium chloride and CEBPα might play important regulatory roles in let-7a2 gene expression in A549 cells.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Medical School of Shandong University, 44 Wenhua West Road, Jinan 250012, China.
authors.forename: Hengyun
authors.initials: H
authors.lastname: Guan
authors.forename: Pengju
authors.initials: P
authors.lastname: Zhang
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Ju
authors.initials: J
authors.lastname: Zhang
authors.forename: Zhaoqin
authors.initials: Z
authors.lastname: Huang
authors.forename: Weiwen
authors.initials: W
authors.lastname: Chen
authors.forename: Zhaobo
authors.initials: Z
authors.lastname: Chen
authors.forename: Nana
authors.initials: N
authors.lastname: Ni
authors.forename: Qingwei
authors.initials: Q
authors.lastname: Liu
authors.forename: Anli
authors.initials: A
authors.lastname: Jiang
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D003907:Dexamethasone / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D018021:Lithium Chloride / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D035683:MicroRNAs; C487931:mirnlet7 microRNA, human; D014212:Tretinoin; D003907:Dexamethasone; D008156:Luciferases; D018021:Lithium Chloride