3′ Noncoding Region Construction of GHR Gene-luciferase Report Vector and Valuation

Abstract

To analyze miR-139 target sites in 3′ UTR of GHR gene in dairy cow mammary gland, a GHR 3′ UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3′ UTR was predicted by Target Scan 5.1 software, 3′ UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe I/Hind III modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3′ UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe I/Hind III digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3′ UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR-139.