Construction of mouse DUSP1 3'-UTR dual luciferase reporter system and its function identification / 解放军医学杂志

ABSTRACT

Objective To construct mouse dual specificity phosphatase-1 (DUSP1) 3'-untranslated region (3'-UTR) dual luciferase reporter system, and to identify its function. Methods The total RNA of RAW264.7 murine macrophage cells was extracted and reverse-transcribed into cDNA. The full length of mouse DUSP1 3'-UTR fragment was amplified by PCR and sub-cloned to the immediate downstream of luciferase cDNA in pGL3-control luciferase expression plasmid to obtain the recombinant plasmid pGL3-Luc-DUSP1- 3'UTR (LuDP). LuDP was then verified by PCR, restriction endonuclease and DNA sequencing analysis. Plasmid LuDP was transiently co-transfected into NIH3T3 cells with internal control plasmid pRL-TK, and the effects of DUSP1 3'-UTR on the expression of attached luciferase gene was analyzed with dual luciferase reporter assay system 48 hours after transfection. Both the luciferase expression module from LuDP and renilla luciferase expression module from pRL-TK were co-subcloned into pLenti6-TR vector to construct dual luciferase reporter plasmid LuDP/RL. The recombinant plasmid LuDP/RL was co-transfected with pcDNA3.1-HuR-FLAG (the eukaryotic expression plasmid of binding protein HuR) and miRNA mimics mmu-miR-101a into NIH3T3 cells respectively. The effects of HuR and mmu-mi-R101a on the expression of DUSP1 3'-UTR attached luciferase gene were also analyzed with dual luciferase reporter assay system. Results The dual luciferase reporter plasmid LuDP/RL was successfully constructed. The mouse DUSP1 mRNA 3'-UTR significantly down-regulated the expression of attached luciferase gene in NIH3T3 cells (0.14 ±0.01 fold, P<0.01). The co-transfected HuR up-regulated the expression of luciferase in LuDP/RL plasmid (1.40 ± 0.20 fold, P<0.05), and co-transfected mmu-miR-101a down-regulated the expression of LuDP/RL (0.57 ± 0.18 fold, P<0.05). Conclusion The constructed dual luciferase reporter plasmid LuDP/RL can be used to analyze the regulatory mechanism of mouse DUSP1 gene expression controlled by post-transcriptional regulatory elements.