Figures
- FIGURE 1
Flow diagram of the systematic research method for detecting matching microRNAs (miRNAs) in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). The upper part of the flow diagram shows the number of papers reviewed and the detailed list of excluded manuscripts. The lower part shows the number of included papers and the remaining part of those after excluding those related to miRNAs not overlapping for SSc and IPF.
- FIGURE 2
Fibroblast responses to changes in miR-21-5p and miR-29 family levels. miR-21-5p and miR-29 family members can act synergistically to potentiate fibroblast functions in fibrotic conditions. The figure shows complex but established relationships supported by the literature review. Above the horizontal line is the activation of a profibrotic fibroblast in both idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). Below the line represents the normal state. Highlighted on the right surrounding the abstract fibroblast is the mechanism by which both miRNAs can regulate transforming growth factor (TGF)-β release from its latency-associated peptide (LAP) by stimulating integrin expression and collagen synthesis. The right-hand fibroblast image also shows the effect of miRNAs 21-5p and the 29 family on α-SMA expression. Small mother against decapentaplegic protein (SMAD)3 has a pivotal role in the maintenance of the loop of this finely regulated system: SMAD3 stimulates the secretion of miR-21-5p, which blocks the inhibitory effect of SMAD7 on SMAD3; in addition, SMAD3 reduces miR-29 family levels, which reinforces SMAD3 activation.
Tables
- TABLE 1
Overview of putative functions of the most relevant microRNAs (miRNAs) overlapping in systemic sclerosis and idiopathic pulmonary fibrosis
Putative functions miR-29a-3p Collagen gene expression – apoptosis miR-29b-3p Collagen gene expression miR-29c-3p ECM-related genes expression miR-21-5p EMT – collagen expression – SMAD7 miR-92a-3p MMP-1 expression miR-26a-5p EMT – collagen expression let-7d-5p EMT The table shows candidate functions for each major overlapping miRNA in systemic sclerosis and idiopathic pulmonary fibrosis, and provides an overview of the specific functions in fibrogenesis regulated by a specific miRNA. ECM: extracellular matrix; SMAD7: small mother against decapentaplegic protein 7; MMP-1: matrix metalloproteinase 1; EMT: epithelial–mesenchymal transition.
- TABLE 2
Overlapping microRNAs (miRNAs) in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF)
miRNAs Specimen Method Target mRNA Design Subjects n [Ref.] 29a-3p SSc and normal dermal fibroblasts miR-29a-3p mimic/antimir Bcl-2 Bax Case–control dcSSc=10, controls=10 [18] 29a-3p, 29b-3p, 29c-3p, pre-mir-29a, pre-mir-29b Dermal fibroblasts qPCR, pre-mir29a/b mimic N/A Cross-sectional Controls=9 [17] 29a-3p, 29b-3p, 29c-3p, pre-mir-29a, pre-mir-29b, pre-mir-29c SSc and normal skin biopsy; dermal fibroblasts qPCR, pre-miR29a/b/c mimic/antimir COL1A1, COL3A1 Case–control SSc=12, controls=11 [15] 29c-3p, pre-mir-29c IPF and normal lung biopsy; primary mesenchymal cells and controls; pulmonary fibroblasts qPCR, miR-29c-3p overexpression/knockdown lentiviral transfection N/A Case–control IPF=11, controls=10 [22] 29c-3p IPF and normal lung biopsy; pulmonary fibroblasts; decellularised primary human lung; ECM qPCR, miR-29c-3p lentiviral transfection COL6A2, LAMA2, COL4A2, COL1A2, LAMC1, COL4A1, COL1A1, COL5A1, NID1, MFAP2 Case–control IPF=5, controls=5 [23] 21-5p SSc and normal skin biopsy; dermal fibroblasts qPCR, in situ hybridisation, miR-21-5p mimic/antimir FN1, ACTA2, COL1A1, COL1A2 Case–control dcSSc=4, lcSSc=2, controls=4 [28] 9-5p, 10a-5p, 15a-5p, 21-5p, 31-5p, 125a-5p, 125b-5p, 137-3p, 181a-5p, 203a-3p, 218-5p, 381-3p, 410, let-7a-5p, let-7b-5p, let-7c Serum Array, initial qPCR for miR-146a-5p, 155-5p, let-7a-5p, 181a-5p, 454-5p, let-7b-5p, 28-3p and miR-885-5p; confirmatory qPCR for miR-21-5p, 199a-5p, 200c-3p, 31-5p, let-7a-5p and let-7d-5p N/A Case–control Rapidly progressive=12, slowly progressive IPF=12, controls for profiling=12; rapidly progressive IPF=20, slowly progressive IPF =24, controls=20 for qPCR confirmation [24] 21-5p, 155-5p Serum qPCR N/A Case–control IPF=65, controls=65 [30] 21-5p IPF and normal lung biopsy; MRC-5 and IMR-90, HEK-293 cells Northern blotting, in situ hybridisation, miR-21-5p mimic/antimir N/A Case–control IPF=8, controls=8 [31] 21-5p IPF and normal lung biopsy; human alveolar type 2 cells qPCR, in situ hybridisation N/A Case–control IPF=3, controls=3 [32] 21-5p, 29a-3p, 92a-3p, 101-3p, 106a-5p, 142-3p, 155-5p, 181b-5p-5p, 184-3p, 223-3p, 342-3p, 409-3p, let-7c Serum qPCR N/A Cross-sectional dcSSc=32, lcSSc=63 [21] 17-5p, 20a-5p, 29b-3p, 92a-3p, 106a-5p, 142-3p, 145-5p, 146b-5p, 181b-5p-5p, 221-3p, 223-3p, 342-3p Serum qPCR N/A Case–control dcSSc=41, lcSSc=79, SLE=29, controls=40 [20] 26a-5p, 30a-5p, 30b-5p, 30d-5p, 92a-3p, 101-3p, 125a-5p, 126-3p, 184-3p, 203a-3p, 218-5p, 222-3p, 375-3p, let-7d-5p, let-7g-5p IPF and normal lung biopsy; pulmonary fibroblasts qPCR, miR-30a-5p, 30d-5p, 92a-3p mimic/antimir WISP1 Case–control IPF=8, controls=7 [37] 92a-3p SSc and normal skin biopsy; dermal fibroblasts; serum qPCR, miR-92a-3p mimic N/A Case–control dcSSc=23, lcSSc=38, SSD=12, DM=7, SLE=7, controls=18 [34] 17∼92 cluster (17-5p, 18a-5p, 19a-3p, 19b-3p, 20a-5p, 92a-3p) IPF and COPD lung biopsy; normal and IPF pulmonary fibroblasts qPCR, in situ hybridisation, miR-17∼92 mimic/antimir N/A Case–control >80% FVC IPF+COPD=7, 50–80% FVC IPF+COPD=8, <50% FVC IPF+COPD=9, controls=10 [38] 26a-5p, 27b-3p, 30a-5p, 30d-5p, 92a-3p, 125a-5p, 125b-5p, 140-5p, 145-5p, 197-3p, 214-3p, 377-3p, 381-3p, 486-5p, let-7g-5p Skin biopsy Array, qPCR let-7g-5p, 206-3p, 125b-5p N/A (identified by computational prediction algorithms) Case–control SSc=3, controls=3 [36] 21-5p, 29b-3p, 31-5p, 145-5p, 146a-5p, 503-5p SSc and normal skin biopsy; dermal fibroblasts Array, qPCR for miR-21, 31, 503, 146, 145, 29b SMAD7, SMAD3 COL1A1 Case–control dcSSc=5, lcSSc=2, controls=7 [16] 30a-3p SSc and normal skin; rheumatoid arthritis and normal synovial biopsy; dermal fibroblasts, fibroblasts-like synoviocytes qPCR, miR-30a-3p mimic/antimir BAFF Case–control FLS: RA=5, controls=5;
DF: SSc=4, controls=3[50] 9-5p, 10a-5p, 15a-5p, 17-5p, 18a-5p, 20a-5p, 21-5p, 22-3p, 26a-5p, 92a-3p, 93-5p, 96-5p, 100-5p, 101-3p, 103a-3p, 106b-5p, 125a-5p, 126-3p, 130a-3p, 132-3p, 133b-3p, 134, 137-3p, 141-3p, 142-3p, 142-5p, 146a-5p, 155-5p, 181a-5p, 182-5p, 205-5p, 210, 214-3p, 222-3p, 223-3p, 301a-3p, 302c-3p, 345-5p, 370, 375-3p, 424-5p, 520g, let-7a-5p, let-7b, let-7c, let-7d-5p, let-7g-5p Serum; dermal fibroblasts; skin tissues Array, qPCR for let-7a-5p, in situ hybridisation, let-7a mimic/antimir N/A Case–control Serum: dcSSc=20, lcSSc=19, LSc=39, SLE=8, DM=8, controls=17;
skin tissues: SSc=7, LSc=7, keloid=5, controls=7[43] 19a-5p, 26a-5p, 30b-5p, 106b-5p, 181a-5p, 181b-5p, 203a-3p, 205-5p, 302c-3p, 409-3p, 410 let-7a-5p, let-7b-5p, let-7c, let-7d-5p SSc and normal skin biopsy; dermal fibroblasts; serum qPCR, miR-30b-5p mimic PDGFR-β, COL1A2, ACTA2 Case–control dcSSc=18, lcSSc=32, controls=24 [45] 10a-5p, 15a-5p, 17-5p, 18a-5p, 20a-5p, 21-5p, 93-5p, 96-5p, 101-3p, 103a-3p, 106b-5p, 130a-3p, 132-3p, 133b-3p, 134, 137-3p, 141-3p, 142-5p, 142-3p, 155-5p, 182-5p, 210, 301a-3p, 370, 424-5p, 520g, let-7a-5p, let-7b-5p, let-7c, let-7d-5p, let-7g-5p SSc and normal skin biopsy; dermal fibroblasts Array, qPCR for miR-150, in situ hybridisation, miR-150 mimic N/A Case–control Serum: dcSSc=20, lcSSc=20, DM=5, SLE=5, controls=20;
skin tissues: dcSSc=5, controls=5[46] 1-3p, 9-5p, 10a-5p, 15a-5p, 17-5p, 18a-5p, 20a-5p, 21-5p, 22-3p, 26a-5p, 92a-3p, 93-5p, 96-5p, 100-5p, 101-3p, 103a-3p, 106b-5p, 125a-5p, 126-3p, 128-3p, 130a-3p, 132-3p, 133b-3p, 134, 137-3p, 142-5p, 142-3p, 155-5p, 181a-5p, 182-5p, 205-5p, 210, 214-3p, 218-5p, 222-3p, 223-3p, 301a-3p, 302c-3p, 370, 375-3p, 424-5p, 520g, let-7a-5p, let-7b-5p, let-7c, let-7d-5p, let-7g-5p SSc and normal skin biopsy; dermal fibroblasts Array, qPCR for miR-196a, miR-196a mimic/antimir N/A Case–control Serum: dcSSc=20, lcSSc=20, controls=25;
skin tissues: SSc=5, TGF-β-stimulated=5, controls=5[49] 1-3p SSc and normal dermal fibroblasts Array N/A Case–control Skin tissues: dcSSc=6, controls=8 [54] 142-3p Serum qPCR N/A Case–control dcSSc=23, lcSSc=38, SSD=12, controls=20, SLE=8, DM=8 [51] 9-5p IPF and normal lung biopsy; human fetal lung fibroblasts; human mesothelial cells Array, qPCR for miR-9-5p, in situ hybridisation, pre-miR-9-5p mimic/antimir TGFBR2, NOX4, ACTA2, COL1A1, FN1 Case–control IPF=7, controls=3 [55] 31-5p, 424-5p TGF-β1 stimulated and normal human lung epithelial cells (A549) Array, miR-424-5p, 1224-5p and 23b-3p overexpression/knockdown lentiviral transfection N/A Case–control Cells [57] 96-5p IPF and normal lung biopsy; pulmonary fibroblasts qPCR, in situ hybridisation, miR-96-5p mimic/antimir FoxO3a, p27, p21, BIM Case–control IPF=8, controls=8 [53] 210 IPF and normal lung biopsy; lung
mesenchymal cellsqPCR, in situ hybridisation, miR-210 overexpression/knockdown lentiviral transfection N/A Case–control IPF=7, controls=6 [48] 26a-5p Human lung epithelial cells (A549) Array (downloaded from Gene Expression Omnibus database), miR-26a-5p mimic/antimir Human genes annotated in the biological process of “EMT” were downloaded from the Gene Ontology database (GO:0001837) Case–control Cells [41] 26a-5p, 30a-5p, 30b-5p, 133b-3p IPF and normal lung biopsy; human fetal lung fibroblasts Array, miR-26a-5p mimic COL1A1, COL3A1 Case–control IPF=8, controls=10 cells [40] let-7d-5p Human fetal lung fibroblasts, human lung fibroblasts, human fetal
foreskin fibroblastslet-7d-5p overexpression lentiviral transfection PAI-1, HMGA2, ACTA2, CDH2, ID1, ID2, SLUG, FSP-1, FN1, KRT19 Case–control Cells [47] 140-5p Human type II alveolar epithelial cells (A549) qPCR, miR-140-5p mimic/antimir Case–control IPF=5, controls=5 cells [56] 1-3p, 10a-5p, 17-5p, 27b-3p, 30a-3p, 30a-5p, 30b-3p, 30d-5p, 126-3p, 133b-3p, 181a-5p, 184-3p-3p, 203a-3p, 210, 222-3p, 370, 375-3p, 377-3p, 381-3p, 409-3p, 410, 520g, let-7d-5p IPF and normal lung biopsy; IPF, TGF-β-stimulated and normal human lung fibroblasts Array, miR-154-5p mimic/antimir MEG3, DKK2, DIXDC1, PPP2CA, FZD 4/5/6, LRP, KREMEN1, β-CATENIN, WISP1 Case–control IPF=13, controls=12; IPF=32, controls=28 for qPCR confirmation [44] 15a-5p, 17-5p, 18a-5p, 19a-5p, 20a-5p, 21-5p, 22-3p, 27b-3p, 29a-3p, 29b-3p, 29c-3p, 30a-5p, 30b-5p, 92a-3p, 93-5p, 96-5p, 100-5p, 101-3p, 103a-3p, 106b, 125a-5p, 126-3p, 128, 130a, 140-5p, 141, 142-3p, 142-5p, 155, 181b-5p, 210, 222-3p, 223-3p, 302c-3p, 424-5p, let-7c, let-7d-5p IPF and normal lung biopsy, IPF and normal lung fibroblasts Array N/A Case–control Rapidly progressive IPF=9, slowly progressive IPF=6, controls=10 [29] 26a-5p, 30a-3p, 30b-5p, 30d-5p, 92a-3p, 125a-5p, 126-3p, 132-3p, 134, 155-5p, 182-5p, 184-3p, 197-3p, 203a-3p, 205-5p, 214-3p, 409-3p, let-7d-5p IPF and normal lung tissues, A549 cells Array, qPCR for 26a-5p, 30a-3p, 30b-5p, 30d-5p, 92a-3p, let-7d-5p mimic/inhibitor HMGA2, CDH2, VIM, ACTA2 Case–control IPF=10, controls=10 cells [39] 130a-3p, 142-5p BAL fluid samples; IPF and normal macrophages qPCR N/A Case–control IPF=9, control=7 [52] 29a-3p SSc and normal skin biopsy; dermal fibroblasts qPCR, mir-29a mimic COL1A1, TIMP1, TAB1 Case–control SSc=4–6, controls=4–6 [19] 130b-3p SSc and normal skin biopsy; dermal fibroblasts qPCR, miR-130b-3p mimic/antimir COL1A1, COL1A2, FN1, ASMA Case-control SSc=14, controls=14 [58] 130b-3p IPF and normal lung biopsy; human primary type II alveolar epithelial and lung fibroblasts qPCR, miR-130b-3p mimic/antimir IGF1 Case–control IPF=4, controls=3 [59] 26a-5p, let-7d-5p Human alveolar basal epithelial cells qPCR, miR-26a-5p mimic/antimir LIN28B Case–control IPF=106, controls=50 (microarray data set of GSE32538) [42] 29b-3p IPF lung biopsy qPCR, miR-29b-3p mimic COL1A1, COL3A1 Case–control IPF=16 [25] The table describes all the manuscripts that reported overlapping miRNAs between SSc and IPF. The manuscripts have been categorised according to the significance, specimen analysed, miRNAs detection method employed and, where described, the mRNA target. Moreover, the design of the study and the number of subjects involved in each study, together with the reference, are reported. Bcl-2: B-cell lymphoma 2; BAX: bcl-2-like protein 4; dcSSc: diffuse cutaneous SSc; COL1A1, COL1A2, etc.: collagen, type I, alpha 1, collagen, type I, alpha 2, etc.; ECM: extracellular matrix; LAMA2: laminin, alpha 2; LAMC1: laminin, gamma 1; NID1: nidogen 1; MFAP2: microfibrillar-associated protein 2; FN1: fibronectin 1; ACTA: alpha smooth muscle actin; lcSSc: limited cutaneous SSc; MRC-5: Medical Research Council cell strain 5; HEK-293: human embryonic kidney cells 293; SLE: systemic lupus erythematosus; WISP1: WNT1-inducible-signalling pathway protein 1; SSD: scleroderma spectrum disorders; DM: dermatomyositis; COPD: chronic obstructive pulmonary disease; N/A: not available; FVC: forced vital capacity; SMAD: small mother against decapentaplegic protein; BAFF: B-cell activating factor; FLS: fibroblast-like synoviocytes; RA: rheumatoid arthritis; DF: dermal fibroblasts; LSc: localised scleroderma; PDGFR-β: platelet-derived growth factor receptor β; TGF-β: transforming growth factor-β; NOX4: NADPH oxidase 4; FoxO3a: Forkhead box O3; BIM: Bcl-2-like protein 11; EMT: epithelial–mesenchymal transition; PAI-1: plasminogen activator inhibitor-1; HMGA2: high-mobility group AT-hook 2; CDH2: cadherin-2; ID1–ID2: DNA-binding protein inhibitor 1–2; SLUG: protein snail homolog 2; FSP-1: fibroblast-specific protein 1; KRT19: keratin 19; MEG3: maternally expressed 3; DKK2: Dickkopf WNT signaling pathway inhibitor 2; DIXDC1: DIX domain containing 1; PP2CA: protein phosphatase 2CA; FZD4-5-6: frizzled-4 frizzled-5 frizzled-6; LRP: lipoprotein receptor-related protein; KREMEN: Kringle-Containing Protein Marking the Eye and the Nose; VIM: vimentin; BAL: bronchoalveolar lavage; TIMP: tissue inhibitor of metalloproteinase 1; TAB1: TGF-β activated kinase 1/MAP3K7 binding protein 1; ASMA: a smooth muscle actin; IGF: insulin-like growth factor.
- TABLE 3
Major overlapping miRNAs in systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF)
SSc IPF Fibroblasts Serum expression Skin expression Fibroblasts Serum expression Lung expression Expression Effect Expression Effect 29a-3p ↓ Mimic induces apoptosis, abrogates collagen expression, increases collagen degradation ↑ in RNP+ compared to ACA+ ↓ ↓ 29b-3p ↓ Mimic abrogates collagen expression ↑ ↓ ↓ 29c-3p ↓ Mimic abrogates ECM expression ↓ ↓ Overexpression abrogates collagen expression ↓ 21-5p ↔ ↑ in RNP+ compared to ACA+ ↑ Knockdown abrogates collagen expression and EMT ↑ in rapid progressive disease - associated with worse FVC ↑ 92a-3p ↔ Overexpression induces collagen degradation ↔ in SSc and in SSc-ILD ↔ ↔ 26a-5p ↓ ↔ Overexpression abrogates collagen expression and EMT ↓ let-7d-5p ↓ ↓ ↓ Overexpression reduces EMT ↓ in slowly and rapid progressive disease ↓ The table compares the expression levels and the effects of major overlapping miRNAs in SSc skin and fibroblasts with those observed in IPF lung and fibroblasts. Circulating serum levels for SSc and IPF patients are also reported. RNP+: SSc patients with anti-U1 ribonucleoprotein antibodies; ACA+: SSc patients with anticentromere antibodies; ECM: extracellular matrix; EMT: epithelial–mesenchymal transition; FVC: forced vital capacity; SSc-ILD: systemic sclerosis-associated interstitial lung disease. ↑: high levels; ↓: low levels; ↔: controversial results.
