萤光素酶报告基因方法分析miRNA-23a的靶基因.doc

摘要：我们已经发现，miRNA-23a参与调节骨骼肌的分化发育。在此过程中miRNA-23a作用的分子机制目前还不清楚。芯片研究发现了表达受到miRNA-23a下调的系列基因，包括TOP1及CNOT2。本文分别将TOP1及CNOT2基因的3’-UTR区插入荧光素酶报告基因载体pGL3-promoter构建重组质粒。与miRNA-23a-pIRES2-promoter共转染HEK 293细胞，结果发现miRNA-23a对于荧光素酶的表达没有抑制作用。由此得出TOP1及CNOT2基因不是miRNA-23a的直接靶基因。

关键词：miRNA-23a；TOP1；CNOT2；HEK293细胞；pGL3-promoter 质粒

Abstract: We have already discovered that miRNA-23a was involved in the regulation of skeletal muscle differentiation and development, the molecular mechanism is not understood completely till now. Several genes down-regulated by miRNA-23a have been revealed by cDNA chip assay data, including TOP1 and CNOT2 .In this report ,we constructed recom binant plasmids by inserting 3’-UTRs of TOP1 or CNOT2 mRNA into the Luciferase reporter vector pGL3-promoter and then co-transfected them into HEK 293 cells with miRNA-23a expression plasmid –pIRES2 –promoter. The result showed that miRNA-23a could not suppress the Luciferase activity. We conclude that TOP1 and CNOT2 are not the direct target genes of miRNA-23a.

Key words: miRNA-23a; TOP1; CNOT2; HEK293 cell; pGL3-promoter plasmid

　　本课题组前期研究发现miRNA-23a可以下调MHC的表达，从而抑制C2C12成肌细胞的分化。 由于miRNA-23a对于调节骨骼肌分化的调节过程可能涉及多个步骤，有必要研究其作用的分子机制。课题组前期通过表达芯片技术筛选出受到miRNA-23a下调， 并通过生物信息学分析其3’-UTR区具有miRNA-23a结合序列的侯选基因TOP1以及CNOT2。本文分别构建TOP1/CNOT2的3’-UTR-pGL3-promoter 报告基因质粒，然后与参考质粒 pRL-TK及miR-23a的表达质粒miR-23a-pIRES2-EGFP质粒共转HEK293细胞，Luciferase活性测定分析表明，miR-23a 与TOP1及CNOT2mRNA的3’-UTR之间不存在相互作用，即TOP1和CNOT2不是miR-23a的直接靶基因。