Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
Tables
MicroRNA Mean S.D. Maximum Value Minimum Value miR-379-5p 0.6540 0.3377 1.7136 0.2418 miR-135a-5p 0.0290 0.0400 0.2792 0.0007 miR-590-5p 1.1699 0.7811 4.4453 0.2338 miR-216a-5p 0.0716 0.0505 0.2085 0.0127 miR-216b-5p 0.0215 0.0197 0.0934 0.0004 miR-146a-3p 0.0089 0.0061 0.0268 0.0022 miR-196a-5p 0.2286 0.8009 6.3672 0.0140 miR-485-5p 0.0031 0.0020 0.0111 0.0009 ↵a The miRNA expression levels were normalized using the method detailed in Lamba et al. (2014).
Data Supplement
- 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).
- Supplemental Tables -
