Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines
Characterization and functional analysis of the human microRNA let-7a2 promoter in lung cancer A549 cell lines
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肺癌 A549 细胞系中人 microRNA let-7a2 启动子的表征和功能分析
DOI：
10.1007/s11033-011-0683-8
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发表时间：
2011-11-01
影响因子：
2.8
通讯作者：
Jiang, Anli
中科院分区：
文献类型：
--
作者：
Guan, Hengyun;Zhang, Pengju;Jiang, Anli
最近的研究表明，microRNA与人类癌症有很强的关联。miRNA let-7在正常肺组织中高度表达，但在肺癌中经常以降低的水平表达。Let-7a 2是let-7家族的成员。到目前为止，对let-7a 2的转录调控知之甚少。我们的研究主要集中在人miRNA let-7a 2启动子在A549细胞系中的特性和功能分析。首先进行5′ RACE（rapidamplifiedofcDNAends），将let-7a 2基因上游2.8kb的片段克隆到pGL 3-basic载体中。利用MatInspector数据库对let-7a 2基因启动子区进行序列分析，发现let-7a 2基因启动子区存在p53、c-Myc、Ras、CEBPα、RORA、RXR、TCF和GR等重要转录因子的结合位点。采用RT-PCR和let-7a靶序列报告质粒转染的方法，检测9-cis-RA、all-trans-RA、氯化锂和地塞米松对A549细胞let-7a 2基因转录水平的影响。结果表明，重组质粒pGL 3-p7 a2可以作为启动子。CEBPα转染或氯化锂处理可下调该片段的启动子活性，而9-cis-RA或all-trans-RA处理可增强该片段的启动子活性。RT-PCR分析和let-7a靶序列报告质粒转染结果表明，9-cis-RA和all-trans-RA均能上调let-7a 2的表达，而氯化锂则下调其表达。以上结果提示9-cis-RA、all-trans-RA、氯化锂和CEBPα对A549细胞let-7a 2基因表达具有重要的调控作用。
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.