标题：Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines
作者：Guan, Hengyun;Zhang, Pengju;Liu, Chang;Zhang, Ju;Huang, Zhaoqin;Chen, Weiwen;Chen, Zhaobo;Ni, Nana;Liu, Qingwei;Jiang, Anli [Aut
作者机构：[Guan, H] Institute of Biochemistry and Molecular Biology, Medical School of Shandong University, 44 Wenhua West Road, Jinan 250012, China;[ Zhang, P] 更多
通讯作者地址：[Jiang, AL]Shandong Univ, Sch Med, Inst Biochem & Mol Biol, 44 Wenhua W Rd, Jinan 250012, Peoples R China.
来源：Molecular biology reports
关键词：cDNA: complementary DNA;dexamethasone: 50-02-2;microRNA;RXR;p53 protein;c-Myc;transcriptional factor;lithium chloride: 7447-41-8;Ras protein;TCF;GR;RORA protein;let-7a2 promoter: expression;upregulation;transcriptional regulation;CEBP alpha;sequence-reporter plasmid;pGL3-p7a2;MatInspector database
摘要：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, CEBP alpha, 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 CEBP alpha 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 CEBP alpha might play important regulatory roles in let-7a2 gene expression in A549 cells.