Supplemental Data For

1 DMD Manuscript # 52126

Supplemental Data for

Drug Metabolism and Disposition

Expression variability of ADME-related microRNAs in human liver:

influence of non-genetic factors and association with gene expression

Jessica K. Rieger, Kathrin Klein, Stefan Winter, Ulrich M. Zanger

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, D-70376 Stuttgart

and University of Tuebingen, Germany (JKR, KK, SW, UMZ)

2 DMD Manuscript # 52126

Supplemental Table 1: Self-designed Taqman assay primers (for, forward; rev, reverse) and probe for mRNA quantification of CYP1A1.

Gene Primer/Probe for: TGGTCAAGGAGCACTACAAAACC CYP1A1 rev: TCATCTGACAGCTGGACATTGG

probe: GATGTGGCCCTTCTC 3 DMD Manuscript # 52126

Supplemental Table 2: Interindividual variability of ADME-related miRNA expression in human liver Coefficient of MiRNA Variabilitya Predicted targetsb variation let-7a 9.82 50% ABCC2, CYP4F2, NR1I2, PPARA, SLC22A3, SLC22A7, SLC2A2

ABCC2, ALDH2, CYP2D6, CYP4F2, NR1I2, PPARA, SLC10A1, SLC22A3, SLC22A7, SLC2A2, let-7b 12.1 54% SREBF2 let-7c 13.3 64% ABCC2, ALDH2, CYP2D6, CYP4F2, NR1I2, PPARA, SLC10A1, SLC22A3, SLC22A7, SLC2A2 let-7d 16.1 69% ABCC2, CYP4F2, NR1I2, PPARA, SLC22A3, SLC22A7, SLC2A2 let-7e 19.5 69% ABCC2, ALDH2, CYP4F2, NR1I2, PON1, PPARA, SLC22A3, SLC22A7, SLC2A6, SREBF2 let-7f 31,298 83% ABCC2, CYP4F2, NR1I2, PPARA, SLC22A3, SLC22A7, SLC2A2, SREBF2 let-7g 10.2 51% ABCC2, CYP3A5,CYP4F2, NR1I2, PPARA, SLC22A3, SLC22A7, SLC2A, UGT2B7 miR-101 46.0 82% AHRR, CYP2C18, CYP2C19, CYP4F2, HMGCR, PPARA, SLC10A1, SLC22A3 miR-106a 14.8 55% ABCG2, AHRR, , CYP3A4, DPYD, PPARA, SLC22A2, SLC22A3, SLC2A2 miR-106b 10.8 55% ABCG2, AHRR, , CYP3A4, DPYD, PPARA, SLC22A2, SLC22A3, SLC2A2 miR-10a 14.9 64% ALDH2, HMGCR, INSIG2, PPARA, RXRA, SLC10A1, SLCO2B1, TPMT, VDR miR-122 19.4 76% ABCG8, CYP3A4, PPARA, SLC22A3, SLC22A7, VDR

ABCB11, ABCC2, AHRR, CYP2D6, HMGCR, HNF4a, PPARA, SLC22A3, SLC22A7, SLCO2B1, miR-125b 8.0 49% VDR AHRR, CYP2B6, CYP2C9, CYP4F2, GSTM1, GSTT1, HNF4a, INSIG1, NR3C1, PPARG, RXRA, miR-130a 29.0 77% SLCO1B1 AHRR, CYP2B6, CYP2C9, CYP4F2, GSTM1, GSTT1, HNF4a, INSIG1, NR3C1, PPARG, RXRA, miR-130b 83.9 108% SLCO1B1 miR-132 116.0 127% ABCG2, CYP3A43, DPYD, INSIG1 PPARA, RXRA, SLC22A3

CYP3A4, CYP3A7, GSTP1, HNF4a, NR0B2, NR1I2, NR1I3, SLC22A2, SLC47A1, SLCO1B3, miR-133a 181.4 139% SREBF2, TPMT, UGT2B7 miR-142-3p 37.1 95% CYP1A2, CYP3A4, NR0B2, NR1I3, NR3C1, PPARA, UGT2B7, VDR

ABCB11, ABCG5, CYP1A2, CYP2B6, CYP2C19, CYP2C9, HNF4a, PPARA, SLC22A3, SLC22A7, miR-143 17.4 85% SLCO2B1, UGT1A1, UGT1A3, UGT1A5 miR-146a 13.1 62% ABCG2, AHRR, CYP2E1, CYP2A43, HNF4a, POR, SLC22A7, SREBF2

AHRR, CYP2B6, CYP3A7, CYP4F2, HNF4a, NR1I2, PPARA, RXRA, SLC2A2, SLCO2B1, UGT1A1, miR-148a 17.5 68% UGT1A3, UGT1A5 AHRR, CYP2B6, CYP3A7, HNF4a, NR1I2, PPARA, RXRA, SLC2A2, SLCO2B1, UGT1A1, UGT1A3, miR-148b 263 98% UGT1A5 ABCG5, ABCG8, CYP1A1, CYP1B1, CYP2A6, CYP3A4, GSTP1, HNF4a, NR3C1, PPARA, RXRA, miR-150 24.3 83% SLC22A2, SLC22A7, SLC22A9, TPMT, UGT1A1, UGT1A3, UGT1A5, VDR AHRR, CYP2B6, CYP3A4, CYP3A7, HNF4a, NR1I2, PGRMC2, PPARA, RXRA, SLC2A2, SLCO2B1, miR-152 13.5 65% UGT1A1, UGT1A3, UGT1A5 miR-15a 1,368 163% CYP1B1, CYP2C9, HNF4a, NR1I3, PPARA, SLC15A2, SLC22A2, SLC22A7 miR-16 22.6 84% CYP1B1, CYP2C9, HNF4a, PPARA, SLC22A2 miR-17 28.1 67% ABCG2, AHRR, CYP3A4, DPYD, PPARA, SLC22A2, SLC22A3, SLC2A2

AHRR, CYP2B6, GSTP1, HNF4a, INSIG1, POR, PPARA, SLC22A3, SLCO2B1, SREBF2, TPMT, miR-185 165 129% VDR ABCC2, ABCG8, AHRR, CYP2B6, CYP3A7, DPYD, HNF4a, NR1I2, NR3C1, PPARA, RXRA, miR-18a 27.7 95% SLC10A1, SLC22A3, SLC22A9 ABCC2, ABCG8, AHRR, CYP2B6, CYP3A7, DPYD, HNF4a, NR1I2, NR3C1, PPARA, RXRA, miR-18b 70.2 109% SLC10A1, SLC22A3, SLC22A9 miR-19a 15.9 55% ABCB1, ABCC2, ABCG2, INSIG1, INSIG2, PPARA, RXRA, SLC22A3 4 DMD Manuscript # 52126

miR-19b 10.4 52% ABCB1, ABCG2, INSIG1, INSIG2, PPARA, RXRA, SLC22A3

CYP1B1, CYP2B6, CYP2C8, CYP3A4, CYP3A7, NR0B2, NR3C1, PPARA, RXRA, SLC15A2, miR-200a 522 192% UGT1A1, UGT1A3, UGT1A5 ABCB11, AHRR, CYP1B1,CYP3A4, CYP3A43, CYP4F2, CYP7A1, NR3C1, PPARA, RXRA, miR-200b 986 254% SLC15A2 ABCB11, AHRR, CYP1B1,CYP3A4, CYP3A43, CYP4F2, CYP7A1, NR3C1, PPARA, RXRA, miR-200c 7,323 399% SLC15A2 ABCB11, ABCG8, AHRR, CYP1A1, CYP1B1, HMGCR, NR1I3, NR3C1, PPARA, RXRA, SLC22A3, miR-204 49.1 79% SLC22A6, SLC22A7, SLC22A9, SLCO2B1, UGT1A1, UGT1A3, UGT1A5 miR-21 41.0 98% AHRR, HMGCR, HNF4a, PPARA, SLC10A1, UGT2B15, UGT2B17

ABCB1, CYP2B6, CYP2C18, GSTM1, HNF4a, NR1I2, NR1I3, NR3C1, POR, PPARA, SLC47A1, miR-214 42.2 94% TPMT, VDR miR-22 1,024 230% ABCB11, AHRR, CYP2A6, INSIG1, NR3C1, PPARA, RXRA, SLC22A3, UGT1A1, UGT1A3 miR-221 60.5 110% ABCG2, AHRR, CYP1B1, CYP7A1, INSIG1, NR1I2, SLC22A3, UGT2B15, VDR miR-223 28.6 86% ABCB1. AHRR, CYP1B1, CYP2E1, , NAT1, PPARA, SLC22A9, SLC47A1, SLCO1B3, TPMT, VDR

ABCG8, AHRR, CYP1A1, CYP1A2, CYP3A4, GSTT1, HNF4a, INSIG1, NR1I2, NR1I3, POR, miR-24 11.7 62% PPARA, PPARG, RXRA, SLC22A2, SLC22A9, SLCO2B1, SREBF2, TPMT ABCC2, CYP2E1, CYP3A4, HNF4A, HNF4a, PPARA, SLC22A9, TPMT, UGT1A1, UGT1A3, miR-26a 6.9 44% UGT1A5 ABCC2, CYP2E1, CYP3A4, HNF4A, HNF4a, PPARA, SLC22A9, TPMT, UGT1A1, UGT1A3, miR-26b 21.3 64% UGT1A5 ABCB1, ABCG5, AHRR, CYP1B1, CYP3A4, CYP3A43, CYP4F2, DPYD, HMGCR, INSIG1, INSIG2, miR-27a 20.8 92% NR3C1, PPARA, PPARG RXRA, SLC47A1 ABCB1, AHRR, CYP1B1, CYP3A4, CYP3A43, CYP4F2, DPYD, HMGCR, INSIG1, INSIG2, NR3C1, miR-27b 27.5 81% PPARA, PPARG, RXRA, SLC47A1 miR-28-3p 9.3 46% CYP1B1, CYP2B6, CYP2C19, CYP2C9, HMGCR, INSIG1, SLC47A1, UGT2B15 miR-28-5p 13.4 74% AHRR, CYP2B6, NR0B2, PPARA, SLC22A3, TPMT, UGT1A1 miR-29a 18.7 88% CYP2B6, HMGCR, HNF4a, INSIG1, NR3C1, PPARA, RXRA, SLC22A7, SLC2A2 miR-31 2,096 297% ABCG2, CYP1B1, NAT1, PPARA, SLC10A1, SLC47A1, SLCO2B1

ABCC2, ABCG2, AHRR, CYP1A1, CYP1B1, CYP2D6, HMGCR, INSIG1, NR1I2, SLC22A7, miR-323-3p 89.2 119% SLC2A2, SLC47A1, TPMT, VDR miR-34a 129 110% AHRR, DPYD, HNF4a, NR1I2, PPARA, PPARG, RXRA, SLC15A2, SLC22A3, SLC22A7

ABCB1, ABCB11, ABCC2, ABCG8, AHRR, CYP3A4, CYP3A7, CYP4F2, PPARA, SLC15A2, miR-455-3p 36.0 57% SLC22A3, VDR miR-455-5p 23.3 71% CYP1A1, CYP1B1, DPYD, POR, PPARA, SLC22A7, SLC47A1, SLCO2B1, UGT2B15

ABCC2, AHRR, CYP2C9, CYP2E1, CYP3A4, CYP3A43, CYP3A7, CYP7A1, DPYD, HMGCR, miR-539 21,216 216% HNF4a, INSIG2, PPARA, RXRA, SLC22A3, SLCO2B1, SREBF2, TPMT AHRR, CYP1B1, CYP3A5, CYP3A7, CYP7A1, HNF4a, INSIG1, INSIG2, NR1I3, PPARA, RXRA, miR-9 212 149% SLC22A3, SLC22A7, SLC2A2, SLCO2B1, TPMT, VDR aratio of maximal and minimal expression value bADME-specific target prediction by three databases (microCosm1, TargetScan2 and miRDB3) compiled by using MIRNA-DISTILLER (Rieger et al., 2011)

1 http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5 2 http://www.targetscan.org/vert_50 3 http://mirdb.org/miRDB 5 DMD Manuscript # 52126

Supplemental Table 3: Non-genetic factors influencing miRNA expression in multivariate median regression

a MiRNA P-value

Sex miR-150 0.017 (male vs female)

Age miR-125b 0.043 (increasing) miR-152 0.008 miR-22 0.039 miR-29a 0.050

CRP miR-130b 0.0003 (normal vs elevated)

Cholestasis miR-21 0.003 (normal vs elevated) miR-34a 0.023 miR-150 0.026 a For each miRNA, a multivariate median regression with 7 nongenetic factors (sex, age, nicotine and alcohol consumption, pre-surgical drug exposure, cholestasis, CRP) was calculated. Only associations with p<0.05 are listed.

6 DMD Manuscript # 52126

Supplemental Figure 1: Influences of non-genetic factors on miRNA expression. A: Spearman correlations of age and miRNAs; B: miRNA expression of miR-130b in patients with elevated CRP levels compared to normal; C: miR-185a expression in patients consuming no alcohol, 1-2 times a week or daily; D: miR-21, miR-130b, miR-34a and miR-132 expression in cholestasic vs noncholestatic livers.

miR-34a miR-200a 15 40

30 10

10 miRNA expression (relative units) expression (relative miRNA miRNA expression (relative units) expression (relative miRNA 0 0 0 20 40 60 80 100 0 20 40 60 80 100 age age

miR-200b miR-21 50 10 45 40 8 35 30 20 6

15 4 10

5 miRNA expression (relative units) expression (relative miRNA 0 units) expression (relative miRNA 0 0 20 40 60 80 100 0 20 40 60 80 100 age age

miR-132 miR-200c 20 300 250 200 18 150 100 20 16

14 15

6 10 4 5

2 miRNA expression (relative units) expression (relative miRNA miRNA expression (relative units) expression (relative miRNA 0 0 0 20 40 60 80 100 0 20 40 60 80 100 age age

7 DMD Manuscript # 52126

miR-130b 15

***

5 miRA expression units) (relative miRA 0 normal CRP elevated CRP

miR-185 16 ** 14 12 10 8

2 miRA expression units) (relative miRA 0 no alcohol 1-2 a week daily

8 DMD Manuscript # 52126

miR-21 miR-130b 11 *** 12 10 *** 9 10

6 8

6 4

miRA expression miRA (relativeunits) miRA expression (relative units) (relative expression miRA

0 0 normal cholestatic normal cholestatic

miR-34a miR-132 14 *** 17 12 *** 10 16 8 5 15 6

4 3

2 2

miRA expression miRA (relativeunits) miRA expression (relative units) (relative expression miRA

0 0 normal cholestatic normal cholestatic