关键词: 荧光素, 转染, piRNA-DQ566704, smad2

Abstract: Objective To investigate the targeted regulatory effect of piRNA-DQ566704 on the smad2 gene verified by dual luciferase reporter technology. 
Methods The 3′UTR sequence of smad2 gene was obtained from mouse hepatic stellate cell(HSC) genome, to synthesize the wild type (WT) and mutant (MUT) gene fragments of smad2 including the piRNA-DQ566704 binding site and construct the wild type vector of smad2 (pGL6-miR-smad2-3′UTR-WT+pRL-TK) and mutant (pGL6-miR-smad2-3′UTR-MUT+pRL-TK). The smad2 wild-type and mutant double luciferase reporter vector were transfected into 293T cells with piRNA-DQ566704 mimic, piRNA-DQ566704 inhibitor and negative control (NC) to detect the relative luciferase activity of each group and observe the effect of piRNA-DQ566704 on smad2 gene expression. The expression of smad2 mRNA and smad2 protein in mouse HSC transfected with piRNA-DQ566704 mimic, inhibitor and mimic NC was verified by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. 
Results The relative luciferase activity of the constructs carrying piRNA-DQ566704 mimic and smad2-3′UTR-WT was significantly decreased (P＜0.01), and there was no inhibitory effect on the relative luciferase activity of the constructs carrying smad2-3′UTR-MUT (P＞0.05). After overexpression of piRNA-DQ566704 in mouse HSC, the expression of smad2 mRNA and smad2 protein in the mimic group was significantly reduced (P＜0.001). 
Conclusion piRNA-DQ566704 can targetedly regulate the expression of smad2 gene; Therefore,the smad2 is a direct target gene of piRNA-DQ566704. 

Key words: fluorescein, transfection, piRNA-DQ566704, smad2