ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2016, Vol. 47 ›› Issue (12): 2398-2404.doi: 10.11843/j.issn.0366-6964.2016.12.009

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Targeting Verification between Bta-GHR and miR-139

JIN Lian-feng,YU Guang-pu,SUN Xia,LI Qing-zhang,GAO Xue-jun,CUI Ying-jun*   

  1. (College of Life Science,Northeast Agricultural University,Harbin 150030, China)
  • Received:2016-04-15 Online:2016-12-23 Published:2016-12-23

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Abstract:

The aim of this study was to construct a dual luciferase reporter plasmid containing the 3′untranslated region(UTR) of bta-GHR, and verify the targeting relationship between miR-139 and bta-GHR. The binding site of miR-139 to bta-GHR were predicted using bioinformatics. The synthetic 3′UTR fragment of bta-GHR was cloned into psiCHECK-2 vector to construct wild type (psiCHECK-2-GHR-W 3′UTR) or mutant type (psiCHECK2-GHR-M 3′UTR) dual luciferase reporter plasmid. Cultured Hela cells were divided into 4 groups, they were co-transfected with miR-139 mimic or negative control and psiCHECK2-GHR-W 3′UTR or psiCHECK2-GHR-M 3′UTR. The relative luciferase activity was detected. The cultured bovine mammary epithelial cells were transfected with miR-139 mimic, negative control or miR-139 inhibitor. Using real-time quantitative PCR (qPCR), we measured the mRNA level of GHR and miR-139 in cow. The results showed that, by digestion of restriction enzymes, the wild type recombinant plasmid (psiCHECK2-GHR-W 3′UTR) and mutant type recombinant plasmid (psiCHECK2-GHR-M 3′UTR) were proved to be successfully constructed. After psiCHECK2-GHR-W 3′UTR and miR-139 mimic co-transfected Hela cells, the luciferase activity was significantly lower than other treatment groups (P<0.05). Using qPCR,we further confirmed that the mRNA level of GHR was down-regulated by miR-139 (P<0.05). The dual luciferase reporter plasmid containing 3′UTR of wild type or mutant type of bta-GHR was constructed successfully; The targeting relationship between miR-139 and bta-GHR was confirmed by dual luciferase assay and qPCR assay.

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