Supplementary Data

SUPPLEMENTARY DATA

Supplementary Figure 1. Cytokine-Cytokine receptor interaction pathway components predicted to be regulated by let-7 miRNAs. Pathway analysis performed using DIANA LAB mirPATH v.3.(1) Predicted let-7 targets are indicated by red star symbol.

Supplementary Figure 2. TNF-alpha signaling pathway components predicted to be regulated by let-7 miRNAs. Pathway analysis performed using DIANA LAB mirPATH v.3.(1) Predicted let-7 targets are indicated by red star symbol.

Supplementary Figure 3. NF-Kappa-B signaling pathway components predicted to be regulated by let-7 miRNAs. Pathway analysis performed using DIANA LAB mirPATH v.3.(1) Predicted let-7 targets are indicated by red star symbol

Supplementary Figure 4. Bioinformatic analysis of human and mouse Lin28b 3’UTR sequences for interaction with miRNA. Multiple let-7 recognition sites are present. Targetscan (www.targetscan.org).

Human LIN28B ENST00000345080.4 3' UTR length: 4549

Mouse LIN28B ENST00000345080.4 3' UTR length: 4430

Supplementary Figure 5. Lin28b gene expression in carotid plaque tissue from non-diabetic and diabetic patients (n=4-5, ± SEM). Expression was normalized to 18S for gene expression analysis. *P ≤ 0.05.

LIN28B 40 NS

Relative expression qPCR expression Relative 0 c tic ti e e b b ia a Di n-D o N

Supplementary Figure 6. Lipoxins can restore let-7 levels in SMCs and ECs. Let-7d expression in SMCs pretreated with the ALX/FPR2 antagonist BOC2 (10uM, 30 minutes) followed by LXA 4 (1nM, 30 minutes) and TNF-α (1 ng/ml; 24 hours) stimulation. Expression was normalized to U87 for miRNA analysis (n=3, ± SEM). *P ≤ 0.05.

Let-7d

7 * 6 5 4 NS 2

Relative expression qPCR expression Relative 0 d O C A e O LX F LX N B T treat DMS A + F Un TN A + LX + BOC F N T

Supplementary Figure 7. Expression levels of genes associated with ECM and fibrosis in aortic tissue from C57BL/6 mice that received a single tail-vein injection of let-7d mimic or miR-NC for 24 hours (n=6, ± SEM). Expression was normalized to 18S for gene expression analysis and U87 for miRNA analysis. *P ≤ 0.05.

4 miR-NC Let-7d mimic 3

2 * 1

Relative expression qPCR expression Relative 0 1 AI1 A1 MP2 P 3 4A M MMP9 MMP12 COL1A1 COL COL

Supplementary Figure 8. Flow diagram outlining the methodology for assessing secreted cytokine responses from human carotid plaque biopsies following transfection with scrambled miRNA mimic or let-7d mimic for 24 hours.

Human Plaque Studies Carotid Endarterectomy (Recruited from St. Vincent’s University Hospital, Dublin)

Plaque - Histological assessment

Plaque dissection, Culturing, Transfection (24 hours)

Let-7d mimic delivery (Untreated, Control mimic, Let-7d mimic)

RNA extraction – Collect plaque assess let-7d levels in supernatant – each plaque secreted proteome (confirm transfection) analysis Let-7d Pooling Strategy: 50 * Scr 40 30 • Pool 1 (Scrambled mimic): 300ul per 20 plaque (1200ul final volume). 10 Let-7d Relative expression qPCR expression Relative 0 • Pool 2 (Let-7d mimic): 300ul per plaque (1200ul final volume. 7d mimic Let- Scrambled mimic

Supplementary Figure 9. Transfection of human carotid plaque explants with let-7d mimic modulates inflammatory cytokine release. (A) Raw dot-blots indicating cytokine levels in supernatant collected from human carotid plaques 24 hours after transfection with let-7d mimic or scrambled miRNA mimic (n=4). (B) Information on cytokines measured in Proteome Profiler Human XL Cytokine Array.

A. B.

Coordinate Analyte Entrez Coordinate Analyte Entrezinate Coord Analyte Entrez A1, A2 Reference D9, D10 IFN-γ G9, G10 MCP-3 A3, A4 Adiponectin D11, D12 IGFBP-2 G11, G12 M-CSF A5, A6 ApoA1 D13, D14 IGFBP-3 G13, G14 MIF Scrambled A7, A8 Angiogenin D15, D16 IL-1α G15, G16 MIG A9, A10 ANGPT1 D17, D18 IL-1β G17, G18 MIP-1α/MIP-1β A11, A12 ANGPT2 D19, D20 IL-1ra G19, G20 MIP-3α Control A13, A14 TNFSF13B D21, D22 IL-2 G21, G22 MIP-3β A15, A16 BDNF D23, D24 IL-3 G23, G24 MMP-9 miRNA A17, A18 C5/C5a E1, E2 IL-4 H1, H2 Myeloperoxidase A19, A20 CD14 E3, E4 IL-5 H3, H4 Osteopontin A21, A22 TNFRSF8 E5, E6 IL-6 H5, H6 PDGF-AA A23, A24 Reference E7, E8 IL-8 H7, H8 PDGF-AB/BB B3, B4 TNFSF5, E9, E10 IL-10 H9, H10 Pentraxin B5, B6 CHI3L1, E11, E12 IL-11 H11, H12 PF4 B7, B8 CFD E13, E14 IL-12 H13, H14 RAGE B9, B10 C-Reactive E15, E16 IL-13 H15, H16 RANTES B11, B12 CRP E17, E18 IL-15 H17, H18 RBP-4 B13, B14 Cystatin E19, E20 IL-16 H19, H20 Relaxin-2 Let-7d B15, B16 Dkk-1 E21, E22 IL-17A H21, H22 Resistin B17, B18 DPPIV E23, E24 IL-18 H23, H24 SDF-1α miRNA B19, B20 EGF F1, F2 IL-19 I1, I2 Serpin B21, B22 Emmprin F3, F4 IL-22 I3, I4 SHBG C3, C4 ENA-78 F5, F6 IL-23 I5, I6 ST2 C5, C6 Endoglin F7, F8 IL-24 I7, I8 TARC C7, C8 CD178, F9, F10 IL-27 I9, I10 TFF3 C9, C10 FGF-2 F11, F12 IL-31 I11, I12 TfR C11, C12 FGF-7 F13, F14 IL-32 I13, I14 TGF-α C13, C14 FGF-19 F15, F16 IL-33 I15, I16 Thrombospondin-1 C15, C16 Flt-3 F17, F18 IL-34 I17, I18 TNF-α C17, C18 G-CSF F19, F20 IP-10 I19, I20 uPAR C19, C20 GDF-15 F21, F22 I-TAC I21, I22 VEGF C21, C22 GM-CSF F23, F24 KLK3 J1, J2 Reference D1, D2 GROα G1, G2 Leptin J5, J6 VDBP D3, D4 Somatotropin G3, G4 LIF J7, J8 CD31 D5, D6 HGF G5, G6 Lipocalin-2 J9, J10 TIM-3 D7, D8 ICAM-1 G7, G8 MCP-1 J11, J12 VCAM-1 J23, J24 Negative Control

Supplementary Table 1. In silico prediction of let-7-transcript interactions.

Predicted Let-7 target? Position in the context++ context++ score Pct** GENE (Yes/No) miRNA UTR seed match score* percentile** * IL1B NO MCP1 NO P21 NO P53 NO PCNA NO PDGFR NO ICAM1 NO VCAM1 NO LIN28B mmu-let-7a-1-3p 2337-2343 7mer-1A -0.17 95 N/A LIN28B mmu-let-7a-2-3p 1945-1951 7mer-1A -0.05 61 N/A LIN28B mmu-let-7a-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7a-5p 2409-2415 7mer-m8 -0.35 91 0.91 LIN28B mmu-let-7a-5p 2581-2587 7mer-m8 -0.21 73 0.91 LIN28B mmu-let-7a-5p 3997-4003 7mer-m8 -0.31 87 0.86 LIN28B mmu-let-7a-5p 4185-4191 7mer-1A -0.15 61 0.37 YES LIN28B mmu-let-7b-3p 2337-2343 7mer-1A -0.15 93 N/A LIN28B mmu-let-7b-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7b-5p 2409-2415 7mer-m8 -0.32 88 0.91 LIN28B mmu-let-7b-5p 2581-2587 7mer-m8 -0.23 75 0.91 LIN28B mmu-let-7b-5p 3997-4003 7mer-m8 -0.31 87 0.86 LIN28B mmu-let-7b-5p 4185-4191 7mer-1A -0.17 64 0.37 LIN28B mmu-let-7c-1-3p 1944-1951 8mer -0.03 49 N/A

Position in the context++ context++ score Pct** GENE miRNA UTR seed match score* percentile** * LIN28B mmu-let-7c-2-3p 2337-2343 7mer-1A -0.17 95 N/A LIN28B mmu-let-7c-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7c-5p 2409-2415 7mer-m8 -0.34 90 0.91 LIN28B mmu-let-7c-5p 2581-2587 7mer-m8 -0.21 73 0.91 LIN28B mmu-let-7c-5p 3997-4003 7mer-m8 -0.31 87 0.86 LIN28B mmu-let-7c-5p 4185-4191 7mer-1A -0.15 61 0.37 LIN28B mmu-let-7d-5p 36-43 8mer -0.41 94 0.95 LIN28B mmu-let-7d-5p 2409-2415 7mer-m8 -0.32 87 0.91 LIN28B mmu-let-7d-5p 2581-2587 7mer-m8 -0.2 68 0.91 LIN28B mmu-let-7d-5p 3997-4003 7mer-m8 -0.32 87 0.86 LIN28B mmu-let-7d-5p 4185-4191 7mer-1A -0.19 65 0.37 LIN28B mmu-let-7e-5p 36-43 8mer -0.4 93 0.95

LIN28B mmu-let-7e-5p 2409-2415 7mer-m8 -0.35 91 0.91 LIN28B mmu-let-7e-5p 2581-2587 7mer-m8 -0.21 73 0.91 LIN28B mmu-let-7e-5p 3997-4003 7mer-m8 -0.31 86 0.86 LIN28B mmu-let-7e-5p 4185-4191 7mer-1A -0.15 61 0.37 LIN28B mmu-let-7f-1-3p 2337-2343 7mer-1A -0.12 89 N/A LIN28B mmu-let-7f-2-3p 2337-2343 7mer-1A -0.09 83 N/A LIN28B mmu-let-7f-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7f-5p 2409-2415 7mer-m8 -0.35 91 0.91 LIN28B mmu-let-7f-5p 2581-2587 7mer-m8 -0.21 73 0.91 LIN28B mmu-let-7f-5p 3997-4003 7mer-m8 -0.31 86 0.86 LIN28B mmu-let-7f-5p 4185-4191 7mer-1A -0.15 61 0.37 LIN28B mmu-let-7g-3p 2649-2655 7mer-1A -0.06 42 N/A LIN28B mmu-let-7g-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7g-5p 2409-2415 7mer-m8 -0.33 89 0.91

Position in the context++ context++ score Pct** GENE miRNA UTR seed match score* percentile** * LIN28B mmu-let-7g-5p 2581-2587 7mer-m8 -0.21 73 0.91 LIN28B mmu-let-7g-5p 3997-4003 7mer-m8 -0.31 87 0.86 LIN28B mmu-let-7g-5p 4185-4191 7mer-1A -0.15 61 0.37 LIN28B mmu-let-7i-5p 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7i-5p 2409-2415 7mer-m8 -0.32 88 0.91 LIN28B mmu-let-7i-5p 2581-2587 7mer-m8 -0.27 82 0.91 LIN28B mmu-let-7i-5p 3997-4003 7mer-m8 -0.32 88 0.86 LIN28B mmu-let-7i-5p 4185-4191 7mer-1A -0.17 63 0.37 LIN28B mmu-let-7j 37-43 7mer-1A -0.04 34 N/A LIN28B mmu-let-7j 4185-4191 7mer-1A -0.1 68 N/A LIN28B mmu-let-7k 36-43 8mer -0.4 93 0.95 LIN28B mmu-let-7k 2409-2415 7mer-m8 -0.32 88 0.91 LIN28B mmu-let-7k 2581-2587 7mer-m8 -0.23 75 0.91 LIN28B mmu-let-7k 3997-4003 7mer-m8 -0.31 87 0.86 LIN28B mmu-let-7k 4185-4191 7mer-1A -0.17 64 0.37 Il6 mmu-let-7a-1-3p 302-308 7mer-1A -0.12 90 N/A Il6 mmu-let-7a-5p 312-318 7mer-1A -0.34 90 0.63 Il6 mmu-let-7b-3p 302-308 7mer-1A -0.14 92 N/A Il6 mmu-let-7b-5p 312-318 7mer-1A -0.34 90 0.63 Il6 mmu-let-7c-2-3p 302-308 7mer-1A -0.12 90 N/A Il6 YES mmu-let-7c-5p 312-318 7mer-1A -0.34 90 0.63 Il6 mmu-let-7d-5p 312-318 7mer-1A -0.37 91 0.63 Il6 mmu-let-7e-5p 312-318 7mer-1A -0.34 90 0.63 Il6 mmu-let-7f-1-3p 302-308 7mer-1A -0.14 91 N/A Il6 mmu-let-7f-2-3p 302-308 7mer-1A -0.1 86 N/A Il6 mmu-let-7f-5p 312-318 7mer-1A -0.34 90 0.63

Position in the context++ context++ score Pct** GENE miRNA UTR seed match score* percentile** * Il6 mmu-let-7g-5p 312-318 7mer-1A -0.35 91 0.63

Il6 mmu-let-7i-5p 312-318 7mer-1A -0.34 90 0.63 Il6 mmu-let-7j 312-318 7mer-1A -0.27 94 N/A Il6 mmu-let-7k 312-318 7mer-1A -0.34 90 0.63 PDGFB mmu-let-7a-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7b-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7c-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7d-5p 639-645 7mer-m8 -0.31 86 0.81 PDGFB YES mmu-let-7e-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7f-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7g-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7i-5p 639-645 7mer-m8 -0.32 88 0.81 PDGFB mmu-let-7k 639-645 7mer-m8 -0.32 88 0.81 TGFBR1 mmu-let-7a-2-3p 1160-1166 7mer-1A -0.11 80 N/A TGFBR1 mmu-let-7a-5p 66-73 8mer -0.34 90 0.96 TGFBR1 mmu-let-7a-5p 3696-3702 7mer-1A -0.22 75 0.94 TGFBR1 mmu-let-7b-5p 66-73 8mer -0.34 90 0.96 TGFBR1 mmu-let-7b-5p 3696-3702 7mer-1A -0.22 75 0.94 TGFBR1 mmu-let-7c-1-3p 1160-1166 7mer-1A -0.08 75 N/A TGFBR1 YES mmu-let-7c-5p 66-73 8mer -0.34 90 0.96 TGFBR1 mmu-let-7c-5p 3696-3702 7mer-1A -0.22 75 0.94 TGFBR1 mmu-let-7d-5p 66-73 8mer -0.39 93 0.96 TGFBR1 mmu-let-7d-5p 3696-3702 7mer-1A -0.25 77 0.94 TGFBR1 mmu-let-7e-5p 66-73 8mer -0.34 90 0.96 TGFBR1 mmu-let-7e-5p 3696-3702 7mer-1A -0.22 75 0.94 TGFBR1 mmu-let-7f-5p 66-73 8mer -0.34 90 0.96

Position in the context++ context++ score Pct** GENE miRNA UTR seed match score* percentile** * TGFBR1 mmu-let-7f-5p 3696-3702 7mer-1A -0.22 74 0.94 TGFBR1 mmu-let-7g-3p 921-927 7mer-m8 -0.11 61 N/A TGFBR1 mmu-let-7g-5p 66-73 8mer -0.35 91 0.96 TGFBR1 mmu-let-7g-5p 3696-3702 7mer-1A -0.24 76 0.94 TGFBR1 mmu-let-7g-5p 3135-3146 non-canonical N/A N/A N/A TGFBR1 mmu-let-7g-5p 3135-3146 non-canonical N/A N/A N/A

TGFBR1 mmu-let-7i-5p 66-73 8mer -0.35 91 0.96 TGFBR1 mmu-let-7i-5p 3696-3702 7mer-1A -0.22 75 0.94 TGFBR1 mmu-let-7i-5p 3135-3146 non-canonical N/A N/A N/A TGFBR1 mmu-let-7i-5p 3135-3146 non-canonical N/A N/A N/A TGFBR1 mmu-let-7j 67-73 7mer-1A -0.01 10 N/A TGFBR1 mmu-let-7j 3696-3702 7mer-1A -0.1 67 N/A TGFBR1 mmu-let-7k 66-73 8mer -0.34 90 0.96 TGFBR1 mmu-let-7k 3696-3702 7mer-1A -0.22 75 0.94 TNFRA mmu-let-7d-5p 636-643 8mer -0.36 91 0.96 TNFRA mmu-let-7k 636-643 8mer -0.34 90 0.96 TNFRA mmu-let-7f-5p 636-643 8mer -0.34 90 0.96 TNFRA mmu-let-7g-5p 636-643 8mer -0.35 90 0.96 TNFRA mmu-miR-98-5p 636-643 8mer -0.34 90 0.96 YES TNFRA mmu-let-7i-5p 636-643 8mer -0.34 90 0.96 TNFRA mmu-let-7b-5p 636-643 8mer -0.35 90 0.96 TNFRA mmu-let-7c-5p 636-643 8mer -0.34 90 0.96 TNFRA mmu-let-7e-5p 636-643 8mer -0.33 89 0.96 TNFRA mmu-let-7a-5p 636-643 8mer -0.33 89 0.96

* The context++ score (CS) for a specific site is the sum of the contribution of 14 features (Agarwal et al., 2015)(2): site type; supplementary pairing; local AU minimum distance; sRNA1A*; sRNA1C*; sRNA1G*; sRNA8A*; sRNA8C*; sRNA8G*; site8A*; site8C*; site8G*; 3' UTR length*; SA*; ORF length*; ORF 8mer count*; 3' UTR offset 6mer count*; TA (target site abundance); SPS (seed-pairing stability); PCT (probability of conserved targeting)*

** The context++ score percentile rank is the percentage of sites for this miRNA with a less favourable context++ score.

*** PCT, the probability of conserved targeting as described in Friedman et al., 2009,(3) has been calculated for all highly conserved miRNA families.

Supplementary Table 2. In silico prediction of top ranked miRNA-pathway interactions.

MOUSE HUMAN Pathway miRNA #genes targeted in pathway p-value miRNA #genes targeted in pathway p-value TNF -alpha Pathway mmu -let -7d -5p 24 3.88E -69 hsa -miR -522 -5p 31 6.20E -88 mmu -miR -22 -3p 22 1.95E -62 hsa -miR -16 -5p 31 6.20E -88 mmu -let -7b -5p 20 7.38E -56 hsa -miR -34a -5p 30 1.39E -84 mmu -miR -128 -3p 20 7.38E -56 hsa -let -7a -5p 29 2.97E -81 mmu -miR -17 -5p 19 1.28E -52 hsa -miR -17 -5p 25 3.37E -68 mmu -let -7c -5p 19 1.28E -52 hsa -miR -107 25 3.37E -68 mmu -miR -301b -3p 19 1.28E -52 hsa -miR -103a -3p 24 5.31E -65 mmu -let -7i -5p 19 1. 28E -52 hsa -let -7b -5p 23 7.85E -62 mmu -miR -26a -5p 18 2.05E -49 hsa -let -7d -5p 22 1.09E -58 mmu -miR -322 -5p 17 3.04E -46 hsa -let -7g -5p 22 1.09E -58 Cytokine -Cytokine mmu -let -7d -5p 33 4.04E -71 hsa -miR -34a -5p 40 3.30E -85 Receptor Interaction mmu -miR -27b -3p 28 2.56E -58 hsa -miR -335 -5p 31 1.71E -62 Pathway (PDGF pathway) mmu -let -7b -5p 28 2.56E -58 hsa -let -7a -5p 27 7.76E -27 mmu -miR -27a -3p 28 2.56E -58 hsa -miR -16 -5p 25 4.01E -25 mmu -let -7c -5p 27 8.18E -56 hsa -miR -27a -3p 23 1.71E -23 mmu -let -7i -5p 27 8.18E -56 hsa -miR -522 -5p 22 3.28E -22 mmu -miR -322 -5p 25 7.24E -51 hsa -let -7d -5p 21 5.96E -21 mmu -miR -16 -5p 23 5.26E -46 hsa -miR -26b -5p 21 5.96E -21 mmu -miR -19b -3p 22 1.31E -43 hsa -miR -27b -3p 20 1.02E -20 mmu -miR -17 -5p 21 3.11E -41 hsa -let -7i -5p 19 1.67E -19 NF -kappa B Pa thway mmu -let -7d -5p 19 3.84E -55 hsa -miR -34a -5p 28 7.85E -84 mmu -let -7i -5p 16 3.45E -45 hsa -miR -522 -5p 18 5.66E -50 mmu -miR -27b -3p 15 6.00E -42 hsa -miR -23b -3p 18 5.66E -50 mmu -let -7c -5p 15 6.00E -42 hsa -let -7a -5p 18 5.66E -50

mmu -let -7b -5p 15 6.00E -42 hsa -let -7g -5p 17 9.09E -47 mmu -miR -27a -3p 15 6.00E -42 hsa -miR -16 -5p 17 9.09E -47 mmu -let -7f -5p 14 9.51E -39 hsa -let -7f -5p 16 1.34E -43 mmu -let -7a -5p 14 9.51E -39 hsa -let -7i -5p 15 1.81E -40 mmu -miR -322 -5p 14 9.51E -39 hsa -let -7e -5p 15 1.81E -40 mmu -let -7e -5p 14 9.51E -39 hsa -miR -155 -5p 15 1.81E -40

Supplementary Table 3. Kolmogorov-Smirnov test for normal distribution

FIGURE EXPERIMENTAL DESCRIPTION Kolmogorov -Smirnov test for normality (p -value)* 1A Let-7b, -7d expression in carotid endarterectomy specimens from patients with NA symptomatic and asymptomatic atherosclerosis (n=3-4, ± SEM). 1B Let-7b expression in carotid endarterectomy specimens from non-diabetic and NON -DIABETIC P=0.0839; DIABETIC P>0.1 diabetic patients (n=5-9) 1C Analysis of pro-inflammatory genes in carotid endarterectomy specimens from ICAM (NON -DIABETIC P=0.212); VCAM (NON -DIABETIC non-diabetic and diabetic patients (n=5-9) P=0.144; MCP1 (NON-DIABETIC P=0.2519) 1E Let-7b, -7d expression in aortic tissue isolated from 10 week ApoE -/- control and Let -7b (control P>0.1, diabetic P>0.1) Let -7d (control P>0.1; diabetic mice (n=6) diabetic P>0.1)

2A Gene expression of markers of SMC activation following PDGF treatment (10 TGFBR1 ( Vehicle P=0.004, PDGF P=0.0395 ); P53 ng/ml; 24 hours). (Vehicle P>0.1, PDGF P=0.0978); PDGFRB (Vehicle P>0.1, PDGF P>0.1); PDGF (Vehicle P>0.1, PDGF P>0.1); PCNA (Vehicle P>0.1, PDGF P>0.1); P21 (Vehicle P>0.1, PDGF P>0.1) 2B Expression of let-7 miRNAs in SMCs treated with PDGF (10 ng/ml; 24 hours) Let -7b (control P>0.1, PDGF P>0.1) Let -7d (control P>0.1; PDGF P>0.1) 2C Expression of P21 and PCNA in SMCs treated with the PDGF inhibitor imatinib, NA followed by treatment with PDGF (10 ng/ml; 24 hours) 2D Expression of let-7b in SMCs treated with the PDGF inhibitor imatinib, followed NA by treatment with PDGF (10 ng/ml; 24 hours). 2E Analysis of P21, PCNA, P53 and TGF βR1 gene expression in SMCs transfected NA with let-7b miRNA or control miRNA (miR-NC) followed by PDGF treatment for 24 hours (10 ng/ml). 2F Cell proliferation as measured by cell number count in SMCs transfected with NA let-7b or miR-NC, followed by stimulation with PDGF (10 ng/ml; 0-48 hours). 2H Quantification of crystal violet stained SMC migratory cells transfected with let- NA 7b or miR-NC, followed by stimulation with PDGF (10 ng/ml; 6 hours).

3A Gene expression analysis of markers of vascular inflammation in SMCs in VCAM (Vehicle P>0.1, TNFa P>0.1); IL6 (Vehicle P>0.1, response to TNF-α (1 ng/ml; 24 hours) TNFa P>0.1); ICAM (Vehicle P>0.1, TNFa P>0.1); TNFRA (Vehicle P>0.1, TNFa P>0.1)

3B Expression of let-7 miRNAs in SMCs treated with TNF-α (1 ng/ml; 24 hours) Let -7b (control P>0.1, TNFa P>0.1) Let -7d (control P>0.1 TNFa P>0.1) 3C Analysis of IL-6 and IL-1β gene expression in SMCs transfected with let-7d IL6 (miR -NC P>0.1; miR -NC + TNFa p>0.1; Let -7d P>0.1; Let -7d miRNA or control miRNA (miR-NC) followed by TNF-α treatment for 24 hours (1 + TNFa P>0.1); IL1B (miR-NC P>0.1; miR-NC + TNFa P>0.1; Let- ng/ml). 7d P>0.1; Let-7d + TNFa P>0.1) 3D Luciferase/Renilla ratio results for SMCs cotransfected with let-7d or control NA miRNA (miR-NC) together with an NF-κB activity reporter plasmid, and subsequently stimulated with TNF-α (1 ng/ml; 24 hours). 3F Representative images and quantification of labelled THP1 monocytes adhered NA to SMCs transfected with let-7d or miR-NC, and stimulated with TNF-α (1 ng/ml; 24 hours).

4B Densitometry of Lin28B protein expression in SMCs stimulated with TNF-α (1 NA ng/ml) and PDGF (10 ng/ml) for 1, 6 and 24 hours 4C LIN28B gene expression in aortas from 10 week diabetic and non-diabetic NA ApoE -/- mice (n=4, ± SEM). 4E Gene expression of markers of SMC activation and inflammation following NA transfection with Lin28B siRNA or control siRNA (siRNA-NC), and subsequent TNF-α treatment for 24 hours (1 ng/ml). 4F Gene expression of markers of SMC activation following transfection with NA Lin28B siRNA or siRNA-NC, and PDGF treatment for 24 hours (10 ng/ml). 4G Expression of let-7d in SMCs transfected with Lin28b siRNA or siRNA-NC and NA treated with TNF-α (1 ng/ml; 24 hours) 4H Expression of let-7b in SMCs transfected with Lin28B siRNA or siRNA-NC and NA treated with PDGF (10 ng/ml; 24 hours).

5A Expression analysis of pro-inflammatory genes in ECs in response to TNF-α (1 MCP1 (Vehicle P>0.1, TNFa P>0.1); IL6 (Vehicle ng/ml; 24 hours) P=0.0081, TNFa P=0.0003) ; ICAM (Vehicle P>0.1, TNFa P>0.1); VCAM (Vehicle P>0.1, TNFa P>0.1) 5B Expression of let-7 miRNAs in ECs treated with TNF-α (1 ng/ml; 24 hours). Let -7b (control P>0.1, TNFa P>0.1) Let -7d (control P>0.1; TNFa P>0.1) 5C Gene expression analysis of markers of vascular inflammation in ECs in MCP1 (Vehicle P>0.1, PDGF P>0.1); IL6 (Vehicle P>0.1, response to PDGF (10 ng/ml; 24 hours). PDGF P=0.0003) ; PDGR1(Vehicle P>0.1, PDGF P>0.1)

6A Gene expression of markers of SMC activation following PDGF stimulation (10 P21 (Control P>0.1 / LXA4 P>0.1 / PDGF P>0.1 / ng/ml; 24 hours) and pre-treatment with or without LXA 4; 1nM, 30 minutes). PDGF+LXA4 P>0.1); PCNA (Control P>0.1 / LXA4 P>0.1 / PDGF P>0.1 / PDGF+LXA4 P>0.1); PDGF (Control P>0.1 / LXA4 P>0.1 / PDGF P>0.1 / PDGF+LXA4 P>0.1) 6B Let-7 expression in SMCs following PDGF stimulation (10 ng/ml; 24 hours) and Let-7b (Control P>0.1 / LXA4 P>0.1 / PDGF P>0.1 / pre-treatment with or without LXA 4 (1nM, 30 minutes). PDGF+LXA4 P>0.1); Let-7d (Control P>0.1 / LXA4 P>0.1 / PDGF P>0.1 / PDGF+LXA4 P>0.1) 6C IL-6 expression in SMCs stimulated with TNF-α (1 ng/ml; 24 hours) and pre- IL6 (Control P>0.1 / LXA4 P>0.1 / TNFa P>0.1 / treatment with or without LXA 4 (1nM, 30 minutes). TNFa+LXA4 P>0.1) 6D Let-7 expression in SMCs stimulated with TNF-α (1 ng/ml; 24 hours) and pre- Let-7d (Control P>0.1 / LXA4 P>0.1 / TNFa P>0.1 / treatment with or without LXA 4 (1nM, 30 minutes). TNFa+LXA4 P>0.1) 6E IL-6 expression in ECs stimulated with TNF-α (1 ng/ml; 24 hours) and pre- IL-6 (Control P>0.1 / LXA4 P>0.1 / TNFa P>0.1 / treatment with or without LXA 4 (1nM, 30 minutes). TNFa+LXA4 P>0.1) 6F Let-7 expression in ECs stimulated with TNF-α (1 ng/ml; 24 hours) and pre- Let-7b (Control P>0.1 / LXA4 P>0.1 / TNFa P>0.1 / treatment with or without LXA 4 (1nM, 30 minutes). TNFa+LXA4 P>0.1); Let-7d (Control P>0.1 / LXA4 P>0.1 / TNFa P>0.1 / TNFa+LXA4 P>0.1)

7A Expression levels of let-7d and direct/indirect target genes in isolated C57BL/6 Let-7d (miR-NC P>0.1, Let-7d mimic P=0.0078) ; ICAM aortas transfected ex vivo with let-7d mimic or miR-NC for 5 hours (n=7-10, ± (miR-NC P>0.1, Let-7d mimic P>0.1); IL6 (miR-NC SEM). P>0.1, Let-7d mimic P>0.1); TGFBR1 (miR-NC P>0.1, Let-7d mimic P>0.1) 7B Expression levels of let-7d and direct/indirect targets genes in isolated 10 week Let-7d (miR-NC P<0.1, Let-7d mimic P>0.1) ; ICAM (miR- diabetic ApoE -/- aortas transfected ex vivo with let-7d mimic or miR-NC for 5 NC P>0.1, Let-7d mimic P>0.1); IL6 (miR-NC P>0.1, Let- hours (n=6, ± SEM). 7d mimic P>0.1); TGFBR1 (miR-NC P>0.1, Let-7d mimic P>0.1) 7C Expression levels of let-7d and direct/indirect targets genes in C57BL/6 mice Let-7d (miR-NC P>0.1, Let-7d mimic P>0.1); ICAM (miR- that received a single tail-vein injection of let-7d mimic or miR-NC for 24 hours NC P>0.1, Let-7d mimic P>0.1); IL6 (miR-NC P>0.1, Let- (n=6, ± SEM). 7d mimic P>0.1); TGFBR1 (miR-NC P>0.1, Let-7d mimic P>0.1) *Red font indicates variables that were not normally distributed after Kolmogorov-Smirnov test. Based on these results, appropriated non-parametric tests were applied.

Supplementary Table 4. Summary of statistical tests and P-value results for all experiments*

FIGURE DESCRIPTION STATISTICAL GENE/MIRNA (P -VALUE) TEST 1A Let-7b, -7d expression in carotid endarterectomy T-test Let-7b (0.004); Let-7d (0.047) specimens from patients with symptomatic and asymptomatic atherosclerosis (n=3-4, ± SEM). 1B Let-7b expression in carotid endarterectomy T-test 0.034 specimens from non-diabetic and diabetic patients (n=5-9) 1C Analysis of pro-inflammatory genes in carotid T-test ICAM1 (0.0677); MCP1 (0.0551) endarterectomy specimens from non-diabetic and diabetic patients (n=5-9) 1E Let-7b, -7d expression in aortic tissue isolated from T-test Let-7b (0.048); Let-7d (0.009) 10 week ApoE -/- control and diabetic mice (n=6)

2A Gene expression of markers of SMC activation T-test/ Mann TGFBR1 (*Mann Whitney P=0.0088) ; P53 (0.0002); following PDGF treatment (10 ng/ml; 24 hours). Whitney test* PDGFRB (0.0046); PDGF (0.0005); PCNA (0.0002); P21 (0.0021) 2B Expression of let-7 miRNAs in SMCs treated with T-test Let-7b (0.0013); Let-7d (0.0127) PDGF (10 ng/ml; 24 hours) 2C Expression of P21 and PCNA in SMCs treated with ANOVA P21 (0.0001; PCNA (0.0001) the PDGF inhibitor imatinib, followed by treatment with PDGF (10 ng/ml; 24 hours) 2D Expression of let-7b in SMCs treated with the ANOVA Control vs PDGF (0.0446) PDGF inhibitor imatinib, followed by treatment with PDGF (10 ng/ml; 24 hours). 2E Analysis of P21, PCNA, P53 and TGF βR1 gene ANOVA P21 (0.003); PCNA (0.0019); P53 (0.0035); TGFBR1 expression in SMCs transfected with let-7b miRNA (0.0041) or control miRNA (miR-NC) followed by PDGF treatment for 24 hours (10 ng/ml). 2F Cell proliferation as measured by cell number ANOVA PDGF versus PDGF+LXA4 (0.0118; Vehicle versus PDGF count in SMCs transfected with let-7b or miR-NC, (0.0001) followed by stimulation with PDGF (10 ng/ml; 0-48 hours).

2H Quantification of crystal violet stained SMC ANOVA 0.0331 migratory cells transfected with let-7b or miR-NC, followed by stimulation with PDGF (10 ng/ml; 6 hours).

3A Gene expression analysis of markers of vascular T-test VCAM (0.000013); IL6 (0.00007); ICAM (0.0001); TNFRA inflammation in SMCs in response to TNF-α (1 (0.0187) ng/ml; 24 hours) 3B Expression of let-7 miRNAs in SMCs treated with T-test 0.0039 TNF-α (1 ng/ml; 24 hours) 3C Analysis of IL-6 and IL-1β gene expression in ANOVA IL6 (0.0001); IL-1B (0.023) SMCs transfected with let-7d miRNA or control miRNA (miR-NC) followed by TNF-α treatment for 24 hours (1 ng/ml). 3D Luciferase/Renilla ratio results for SMCs ANOVA 0.0058 cotransfected with let-7d or control miRNA (miR- NC) together with an NF-κB activity reporter plasmid, and subsequently stimulated with TNF-α (1 ng/ml; 24 hours). 3F Representative images and quantification of ANOVA 0.0089 labelled THP1 monocytes adhered to SMCs transfected with let-7d or miR-NC, and stimulated with TNF-α (1 ng/ml; 24 hours).

4B Densitometry of Lin28B protein expression in T-test TNFa 24h (0.0003); PDGF 24h (0.0025) SMCs stimulated with TNF-α (1 ng/ml) and PDGF (10 ng/ml) for 1, 6 and 24 hours 4C LIN28B gene expression in aortas from 10 week T-test 0.0456 diabetic and non-diabetic ApoE -/- mice (n=4, ± SEM). 4E Gene expression of markers of SMC activation and ANOVA IL6 (0.0048); PDGFRB (0.0084) inflammation following transfection with Lin28B siRNA or control siRNA (siRNA-NC), and subsequent TNF-α treatment for 24 hours (1 ng/ml).

4F Gene expression of markers of SMC activation ANOVA PDGFRB (0.0131); TGFBR1 (0.0174) following transfection with Lin28B siRNA or siRNA- NC, and PDGF treatment for 24 hours (10 ng/ml). 4G Expression of let-7d in SMCs transfected with ANOVA 0.0432 Lin28b siRNA or siRNA-NC and treated with TNF- α (1 ng/ml; 24 hours) 4H Expression of let-7b in SMCs transfected with ANOVA 0.0466 Lin28B siRNA or siRNA-NC and treated with PDGF (10 ng/ml; 24 hours).

5A Expression analysis of pro-inflammatory genes in T-test/ Mann MCP1 (<0.0001); ICAM (0.0014); IL6 (*Mann Whitney ECs in response to TNF-α (1 ng/ml; 24 hours) Whitney test* P<0.0001) ; VCAM (<0.0001) 5B Expression of let-7 miRNAs in ECs treated with T-test Let-7b (0.0218); Let-7d (0.0146) TNF-α (1 ng/ml; 24 hours). 5C Gene expression analysis of markers of vascular T-test/ Mann MCP1 (0.0044); IL6 (*Mann Whitney P=0.5007) ; PDGF-R inflammation in ECs in response to PDGF (10 Whitney test* (0.0003) ng/ml; 24 hours).

6A Gene expression of markers of SMC activation ANOVA P21 (0.0042); PCNA (0.0064) following PDGF stimulation (10 ng/ml; 24 hours) and pre-treatment with or without LXA 4; 1nM, 30 minutes). 6B Let-7 expression in SMCs following PDGF ANOVA Let-7b (0.043); Let-7d vehicle versus PDGF (0.0259); Let- stimulation (10 ng/ml; 24 hours) and pre-treatment 7d vehicle versus PDGF+LXA4 (0.0488) with or without LXA 4 (1nM, 30 minutes). 6C IL-6 expression in SMCs stimulated with TNF-α (1 ANOVA 0.0113 ng/ml; 24 hours) and pre-treatment with or without LXA 4 (1nM, 30 minutes). 6D Let-7 expression in SMCs stimulated with TNF-α (1 ANOVA 0.0395 ng/ml; 24 hours) and pre-treatment with or without LXA 4 (1nM, 30 minutes). 6E IL-6 expression in ECs stimulated with TNF-α (1 ANOVA 0.0144 ng/ml; 24 hours) and pre-treatment with or without LXA 4 (1nM, 30 minutes).

6F Let-7 expression in ECs stimulated with TNF-α (1 ANOVA Let-7b (0.0288); Let-7d (0.0318) ng/ml; 24 hours) and pre-treatment with or without LXA 4 (1nM, 30 minutes).

7A Expression levels of let-7d and direct/indirect T-test/ Mann Let-7d (*Mann Whitney test P<0.0001) ; VCAM (0.0084); target genes in isolated C57BL/6 aortas Whitney test* IL6 (0.0115); TGFBR1 (0.0023) transfected ex vivo with let-7d mimic or miR-NC for 5 hours (n=7-10, ± SEM). 7B Expression levels of let-7d and direct/indirect T-test/ Mann Let-7d (*Mann Whitney test P<0.0022) ; ICAM (0.0124); IL6 targets genes in isolated 10 week diabetic ApoE -/- Whitney test* (0.0323); LIN28B (0.0463) aortas transfected ex vivo with let-7d mimic or miR-NC for 5 hours (n=6, ± SEM). 7C Expression levels of let-7d and direct/indirect T-test Let-7d (0.0125); ICAM (0.0328); VCAM (0.0073); MCP1 targets genes in C57BL/6 mice that received a (0.0125); TGFBR1 (0.05) single tail-vein injection of let-7d mimic or miR-NC for 24 hours (n=6, ± SEM). *Red font indicates variables that were not normally distributed based on Kolmogorov-Smirnov test (Supplemental Material, Table 2). Based on these results, appropriated non-parametric tests were applied.

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