Experimental identification of microRNA targets on the 3′ untranslated region of human FMR1 gene
Experimental identification of microRNA targets on the 3′ untranslated region of human FMR1 gene
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DOI：
10.1016/j.jneumeth.2010.04.022
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发表时间：
2010-06
影响因子：
3
通讯作者：
Yong-Hong Yi;Xun-Sha Sun;Jia-Ming Qin;Qi-hua Zhao;W. Liao;Y. Long
中科院分区：
文献类型：
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作者：
Yong-Hong Yi;Xun-Sha Sun;Jia-Ming Qin;Qi-hua Zhao;W. Liao;Y. Long
FMR1基因在中枢神经系统的发育中发挥着重要作用。 FMR1 表达下调会导致脆性 X 综合征。 MicroRNA (miRNA) 可以通过与其 mRNA 靶标碱基配对来抑制基因表达。通过计算机程序分析，5种miRNA：miR-19a、miR-19b、miR-142、miR-302b*和miR-323-3p可能靶向FMR1 3′非翻译区（3′UTR）上的不同位点，但miR-19a和miR-19b共享相同的靶向位点。为了测试这些 miRNA 是否通过与 FMR1 3' UTR 上的 miRNA 靶标相互作用来抑制报告基因表达，我们开发了两种嵌合构建体：一种构建体表达具有 FMR1 3' UTR 或其 miRNA 靶突变的萤火虫荧光素酶，另一种构建体表达与 GFP 融合的 pre-miRNA。两种构建体共转染的荧光素酶检测显示，miRNA、miR-19b、miR-302b* 和 miR-323-3p 可以抑制 HEK-293 细胞中的基因表达，表明这些 miRNA 在调节 FMR1 表达中发挥作用。本研究中使用的构建体可广泛用于识别任何感兴趣基因中的miRNA靶标，这将极大地促进当前理解miRNA生物学功能的进展。
FMR1 gene plays an important role in the development of central nervous system. Down-regulation of the FMR1 expression leads to fragile X syndrome. MicroRNAs (miRNAs) can repress gene expression by base pairing with their mRNA targets. By computer programs analysis, five miRNAs: miR-19a, miR-19b, miR-142, miR-302b* and miR-323-3p potentially target to different sites on the FMR1 3′ untranslated region (3′ UTR), except that miR-19a and miR-19b share the same targeting site. To test whether these miRNAs repress reporter gene expression by interacting with the miRNA targets on the FMR1 3′ UTR, we developed two chimeric constructs: one construct expressing a firefly luciferase with the FMR1 3′ UTR or its miRNA target mutations and the other construct expressing a pre-miRNA fusing with GFP. Luciferase assay co-transfecting with the two constructs showed that the miRNAs, miR-19b, miR-302b* and miR-323-3p could repress gene expression in HEK-293 cells, suggesting a role of these miRNAs in the regulation of the FMR1 expression. The constructs used in this study can be widely used to identify the miRNA targets in any interested genes, which will greatly promote the current progress in understanding the biological function of miRNAs.