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Molecular Pharmacology

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Research ArticleArticle
Open Access

Potential Regulation of UGT2B10 and UGT2B7 by miR-485-5p in Human Liver

Aimee K. Sutliff, Jian Shi, Christy J.W. Watson, Martina S. Hunt, Gang Chen, Hao-Jie Zhu and Philip Lazarus
Molecular Pharmacology December 2019, 96 (6) 674-682; DOI: https://doi.org/10.1124/mol.119.115881
Aimee K. Sutliff
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Jian Shi
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Christy J.W. Watson
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Martina S. Hunt
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Gang Chen
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Hao-Jie Zhu
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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Philip Lazarus
Department of Pharmaceutical Sciences, Washington State University College of Pharmacy and Pharmaceutical Sciences, Spokane, Washington (A.K.S., C.J.W.W., M.H., G.C., P.L.); and Department of Clinical Pharmacy, University of Michigan College of Pharmacy, Ann Arbor, Michigan (J.S., H.-J.Z.)
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    Fig. 1.

    Prediction of miR-485-5p binding to UGT2B10 and UGT2B7 3′UTRs. The miRNA prediction algorithm TargetScan database was used to identify miRNA binding candidates for five UGT2B mRNAs: UGT2B4, 2B7, 2B10, 2B15, and 2B17. miR-485-5p was predicted to bind to the 3′-UTR of UGT2B10; two proposed binding sites for miR-485-5p to the 3′-UTR of UGT2B7 are shown. Numbers listed correspond to the MRE 3′ locations of the translational stop codon for the corresponding UGT.

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    Fig. 2.

    Expression of UGT2B7, 2B10, and 2B15 vs. miR-485-5p in human liver samples. Expression levels of miR-485-5p were quantified using quantitative reverse-transcription PCR and normalized to the geometric mean of miR-152-3p and miR-23b-3p in the same samples. The Y-axis represents the natural log transformed values for miR-485-5p. The X-axis represents the ratio of UGT2B7, 2B10, or 2B15 to the BSA internal standard, transformed to the natural log value.

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    Fig. 3.

    UGT2B 3′-UTR luciferase activity in the presence of miR-485-5p mimics. (A) The pGL3-UGT2B10 3′-UTR luciferase vector (Wild Type) contains 1086 nucleotides of the UGT2B10 3′-UTR including the MRE for miR-485-5p. The pGL3-UGT2B10 3′-UTR MRE deletion vector (Mutant) contains a four-nucleotide deletion within the potential miR-485-5p MRE seed sequence. The two columns on the left-hand side were performed using transfections of the luciferase vector containing the wild-type UGT2B10 3′-UTR; the two columns on the right-hand side were performed using the luciferase vector containing the UGT2B10 3′-UTR with its seed sequence deletion mutation. (B) The pGL3-UGT2B7 3′-UTR luciferase vector contains 251 nucleotides of the UGT2B7 3′-UTR including the MRE for each of the two predicted binding sites for miR-485-5p. The pGL3-UGT2B7 3′-UTR MRE deletion vectors each contain a four-nucleotide deletion within the potential miR-485-5p MRE seed sequence. The two columns on the left-hand side were performed using transfections of the luciferase vector containing the wild-type UGT2B7 3′-UTR; the next six columns on the right-hand side were performed using the luciferase vector containing the UGT2B7 3′-UTR with seed sequence deletion mutations at site 1, site 2, or both sites together, as indicated in the graph. (C) The pGL3-UGT2B15 3′-UTR luciferase vector contains 467 nucleotides of the UGT2B15 3′-UTR. Luciferase reporter vectors were cotransfected into human embryonic kidney 293 cells with the pRL-TK renilla control vector along with 100 nM of miR-485-5p mimic or scrambled miRNA control. Columns represent the mean ± S.D. of at least three independent experiments and are normalized to the scrambled miRNA-transfected control. *P < 0.05; **P < 0.01, all compared with the referent scrambled control miRNA cotransfected with the luciferase vector containing the corresponding wild-type UGT2B 3′-UTR.

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    Fig. 4.

    Glucuronidation activity in the presence of miR-485-5p mimic. (A) UGT2B10 glucuronidation activity against nicotine in HuH-7 cells transfected with 100 nM miR-485-5p mimic or 100 nM of scrambled miRNA control in HuH-7 and Hep3B cells. Columns represent the mean ± S.E. of four independent experiments. (B) UGT2B7 glucuronidation activity against AZT in HuH-7 cells transfected with 100 nM scrambled or miR-485-5p mimic. Columns represent the mean ± S.D. of six independent experiments.

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    TABLE 1

    Quantitative PCR data for miRNA in normal human liver specimensa

    MicroRNAMeanS.D.Maximum ValueMinimum Value
    miR-379-5p0.65400.33771.71360.2418
    miR-135a-5p0.02900.04000.27920.0007
    miR-590-5p1.16990.78114.44530.2338
    miR-216a-5p0.07160.05050.20850.0127
    miR-216b-5p0.02150.01970.09340.0004
    miR-146a-3p0.00890.00610.02680.0022
    miR-196a-5p0.22860.80096.36720.0140
    miR-485-5p0.00310.00200.01110.0009
    • ↵a The miRNA expression levels were normalized using the method detailed in Lamba et al. (2014).

Additional Files

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    • Supplemental Tables -

      Supplementary Table 1 - List of PCR primers used in the current study.

      Supplementary Table 2 - Surrogate peptides used for quantification of UGT2B subfamily members and the internal standard protein bovine serum albumin (BSA).

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Molecular Pharmacology: 96 (6)
Molecular Pharmacology
Vol. 96, Issue 6
1 Dec 2019
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Research ArticleArticle

miR-485-5p Regulation of UGT2B10 and UGT2B7

Aimee K. Sutliff, Jian Shi, Christy J.W. Watson, Martina S. Hunt, Gang Chen, Hao-Jie Zhu and Philip Lazarus
Molecular Pharmacology December 1, 2019, 96 (6) 674-682; DOI: https://doi.org/10.1124/mol.119.115881

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Research ArticleArticle

miR-485-5p Regulation of UGT2B10 and UGT2B7

Aimee K. Sutliff, Jian Shi, Christy J.W. Watson, Martina S. Hunt, Gang Chen, Hao-Jie Zhu and Philip Lazarus
Molecular Pharmacology December 1, 2019, 96 (6) 674-682; DOI: https://doi.org/10.1124/mol.119.115881
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