Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3′-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

Systemic scleroderma (SSc) is a connective tissue disorder that results in fibrosis of the skin and internal organs. Localized scleroderma (LSc) also manifests tissue fibrosis limited to the skin and s.c. tissue, occasionally involving the muscular tissues beneath the cutaneous lesions (1, 2), but the presence of Raynaud’s phenomenon, acrosclerosis, and involvement of internal organs differentiates SSc from LSc (3). Abnormal collagen metabolism (48) and autoimmunity (9, 10) are considered to be fundamental common characteristics of SSc and LSc, and especially, excess collagen production by dermal fibroblasts is thought to be caused by intrinsic activation of TGF-β signaling in both diseases (5, 11).

In contrast, keloids are also characterized by cutaneous fibrosis that invade adjacent healthy tissue. However, keloid is not an autoimmune disease, and it is still controversial whether the collagen production by keloid fibroblasts is also upregulated (1215). Thus, different factors are likely to mediate the tissue fibrosis in SSc/LSc and keloid, but the exact mechanism involved in each fibrotic condition is still unknown.

Recently, microRNAs (miRNAs) have attracted attention for its involvement in the pathogenesis of various human diseases such as immunological disorders, cancers, and metabolic disorders (1618). miRNAs are small noncoding RNAs that bind to complementary sequences in the 3′-untranslated regions (UTRs) of target mRNAs, resulting in inhibiting their translation into protein. Because >1000 of miRNAs have been identified, which corresponds to 1–5% of all genes in the human genome, miRNAs are thought to be the most abundant class of regulators (19). However, little is known about the role of miRNAs in the pathogenesis of SSc and LSc. In the current study, we tried to evaluate the possibility that miRNAs also play some parts in the collagen metabolism and skin fibrosis of SSc/LSc.

Serum samples were obtained from 39 SSc patients in their first visit. These patients were grouped according to the classification system proposed by LeRoy et al. (20): 20 patients had diffuse cutaneous SSc (dcSSc) and 19 had limited cutaneous SSc (lcSSc), as described previously (21). Similarly, 32 patients with LSc were classified into following three subgroups, as described previously (22, 23): 19 patients with morphea (one or a few circumscribed sclerotic plaque), 8 with linear scleroderma (LS, with linear distribution), and 5 with generalized morphea (GM, four or more lesions > 3 cm in diameter and involvement of two or more areas of the body out of seven areas). When patients had both morphea and LS, they were diagnosed as having GM (22). We also included serum samples from other rheumatic diseases: 8 patients with systemic lupus erythematosus (SLE) and 8 with dermatomyositis (DM). Control serum samples were obtained from 17 healthy age-/sex-matched volunteers. Skin specimens were derived from involved skin of 7 SSc, 7 LSc, and 5 keloid patients. These skin samples and seven control skins were collected and fixed in formaldehyde immediately after resections.

Clinical and laboratory data reported in this study were obtained at the time of serum sampling. Institutional review board approval and written informed consent were obtained according to the Declaration of Helsinki.

Human dermal fibroblasts were obtained by skin biopsy from the affected areas of five SSc patients, LSc patients, and healthy donors. All biopsies were performed with institutional review board approval and written informed consent according to the Declaration of Helsinki. Mouse dermal fibroblasts were obtained from the back skin of male BALB/cAJcl mice. All animal experimental protocols in this study were approved by the Committee on the Animal Research at Kumamoto University.

Primary explant cultures were established in 25-cm2 culture flasks in modified Eagle’s medium supplemented with 10% FCS, 2 mM glutamine, and 50 μg/ml gentamicin, as described previously (24). Fibroblasts between the third and sixth subpassages were used for experiments.

Total RNA from cultured cells was extracted using Isogen (Nippon Gene). First-strand cDNA was synthesized by the PrimeScript RT reagent Kit (Takara). Quantitative real-time PCR used primers and templates mixed with the SYBR Premix Ex Taq ΙΙ Kit (Takara). Primer sets for α1(I) collagen and α2(I) collagen were from Takara. DNA was amplified for 40 cycles of denaturation for 5 s at 95°C and annealing for 30 s at 60°C. The transcript levels were normalized to those of GAPDH.

The miRNA isolation from human or mouse skin tissue was performed using miRNeasy FFPE kit (Qiagen). miRNAs were obtained from the total RNA of cultured cells using an RT2 Quantitative PCR-Grade miRNA Isolation Kit (SABiosciences). Serum miRNAs were purified with miRNeasy RNA isolation kit (Qiagen) following the manufacturer’s instructions with minor modification (25). Briefly, 100 μl serum was supplemented with 5 μl 5 fmol/μl synthetic nonhuman miRNA (Caenorhabditis elegans miR-54; Takara) as controls providing an internal reference for normalization of technical variations between samples. After Qiazol solution (1 ml) was added and mixed well, and then, samples were incubated at room temperature for 5 min. Aqueous- and organic-phase separation was achieved by the addition of chloroform. The aqueous phase was applied to RNeasy spin column and RNeasy MinElute spin column. miRNA was eluted from the column with nuclease-free water.

For PCR array, miRNAs were reverse transcribed into first-strand cDNA using an RT2 miRNA First-Strand Kit (SABiosciences). The cDNA was mixed with RT2 SYBR Green/ROX Quantitative PCR Master Mix, and the mixture was added into 96-well RT2 miRNA PCR Array that includes primer pairs for 88 human miRNAs (SABiosciences). PCR was performed on Thermal Cycler Dice (TP800) (Takara) following the manufacturer’s protocol. Threshold cycle (Ct) for each miRNA was extracted using Thermal Cycler Dice Real-Time System version 2.10B (Takara). The raw Ct was normalized using the values of U6.

The Mir-X miRNA First-Strand Synthesis Kit (BD Clontech) was used for cDNA synthesis from miRNAs. Primers for let-7a, cel-miR-54, or U6 (Takara) and templates were mixed with SYBR Advantage Quantitative PCR Premix (BD Clontech). DNA was amplified for 40 cycles of denaturation for 5 s at 95°C and annealing for 20 s at 60°C on Takara Thermal Cycler Dice (TP800). Transcript levels of let-7a were normalized to those of U6 or cel-miR-54 in the same sample.

For the detection of mature, single-stranded form of let-7a was performed using a miScript PCR system (Qiagen): miScript II RT Kit and miScript SYBR Green PCR Kit (26).

Soluble IL-2R and anti-ssDNA Ab were measured with ELISA, as described previously (27, 28).

Human or mouse dermal fibroblasts were washed with PBS twice and lysed in lysis buffer (BioSource International). Aliquots of cell lysates were separated by electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were blocked in blocking One P buffer (Nacalai Tesque) for 1 h and incubated overnight at 4°C with primary Ab for type I collagen–UNLB (Southern Biotechnology Associates). The membranes were washed with TBS and 0.1% TBST, probed with HRP-conjugated secondary Ab for 1 h, and then washed with TBST again. The detection was performed using ECL system (Thermo Scientific), according to the manufacturer’s recommendations. As a loading control, the same membrane was stripped and reprobed with an Ab against β-actin (Santa Cruz Biotechnology).

In situ hybridization was performed with 5′-locked digoxigenin-labeled nucleic acid probes complementary to human mature let-7a and scrambled negative control (Exiqon) (29, 30). Briefly, human tissues were deparaffinized and deproteinized with protease K for 5 min. Slides were then washed in 0.2% glycine in PBS and fixed with 4% paraformaldehyde. Hybridization was performed at 48°C overnight, followed by blocking with 2% FBS and 2% BSA in PBS and 0.1% Tween 20 for 1 h. The probe–target complex was detected immunologically by incubating with a digoxigenin Ab conjugated to alkaline phosphatase acting on the chromogen NBT/5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science) for 48 h. Slides were counterstained with nuclear fast red, and examined under a light microscope (Olympus BX50).

For a purpose to deliver let-7a persistently into mice skin, the mixtures of miRNA and atelocollagen (AteloGene; Koken) were prepared. let-7a oligo was designed, according to previous papers (31, 32) (Hokkaido System Science); mature chain, 5′-UGAGGUAGUAGGUUGUAUAGUU-3′, which corresponds to the mmu-let-7a, and minor chain, 5′-CUAUACAAUCUACUGUCUUUCC-3′, as the passenger strand. They were annealed to each other. Irrelevant control small dsRNA was also obtained from Hokkaido System Science: 5′-AUCCGCGCGAUAGUACGUAUU-3′ and 5′-AATACGTACTATCGCGCGGAT-3′. An equal volume of AteloGene and miRNA solution (40 μM in small interfering RNA buffer included in AteloGene kit) was mixed by rotating for 20 min at 4°C. The mixtures in a 50-μl volume were administered percutaneously into the abdominal cavity of the 8-wk-old male BALB/cAJcl mice (CLEA Japan) under anesthesia. Mice were housed in a specific pathogen-free environment and did not display any evidence of infection or disease.

Bleomycin (Nippon Kayaku) was dissolved in PBS at a concentration of 1 mg/ml and sterilized by filtration as described previously (33, 34). Bleomycin (100 μl) was injected intradermally into the shaved back of the 8 wk-old male BALB/cAJcl mice daily for 4 wk. The back skin, lung, and liver were removed 1 d after final bleomycin injection. The removed tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (4 μm in thickness) were stained with H&E or Masson’s trichrome. Dermal thickness was evaluated by measuring the distance between the epidermal–dermal junction and the dermal–fat junction in H&E sections under 100-fold magnification by two investigators in a blinded manner.

miRNA inhibitors and miScript target protectors for control or let-7a were purchased from Qiagen. Control or let-7a precursors were obtained from Life Technologies. Lipofectamine RNAiMAX (Invitrogen) was used as transfection reagent. For reverse transfection, miRNA inhibitors, miScript target protectors, or miRNA precursors were mixed with transfection reagent and then added when cells were plated, followed by incubation for 48–96 h at 37°C in 5% CO2. Control experiments showed transfection efficiency is >80% (data not shown).

A luciferase reporter plasmid containing α1(I) collagen 3′-UTR was provided by Dr. N. Mitsutake (Nagasaki University, Nagasaki, Japan) (35). A luciferase construct containing α2(I) collagen 3′-UTR was purchased from GeneCopoeia. Lipofectamine 2000 (Invitrogen) was used as transfection reagent. miRNA inhibitors, synthetic oligos, and reporter plasmids mixed with transfection reagent were added when cells were plated, followed by incubation for 48 h at 37°C. Luc-Pair miR luciferase assay (GeneCopoeia) and FilterMax F5 microplate reader (Molecular Devices) were used to analyze the luciferase expression according to the manufacturer’s protocols.

Statistical analysis was carried out with a Student t test for the comparison of two groups and with a Fisher’s exact probability test for the analysis of frequency. ANOVA was used for multiple comparisons. Correlations were assessed by Pearson’s correlation coefficient. All analyses were performed with Statcel software (OMS). The p values < 0.05 were considered significant.

As an initial experiment, to determine which miRNAs are involved in the pathogenesis of SSc/LSc, a mixture of equal amounts of miRNAs from three SSc, three LSc, three keloid, or three normal skins were prepared. We performed miRNA PCR array analysis consisting of 88 miRNAs involved in human cell differentiation and development, and the miRNA expression profile in involved skin of SSc or LSc in vivo was compared with that of normal skin or keloid (the complete dataset is available at the Gene Expression Omnibus microarray data repository, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE43469; Table I).

Table I.
The expression profiles of miRNAs in the skin of systemic scleroderma, localized scleroderma, and keloid as measured by PCR array
Normal ΔCtSSc ΔΔCtLSc ΔCtKeloid ΔΔCtSSc/Normal
Fold ChangeLSc/Normal
Fold ChangeKeloid/Normal
Fold Change
let-7a −2.94 0.24 0.67 −1.01 0.1 0.1 0.3 
let-7b −2.43 −0.07 0.06 −1.02 0.2 0.2 0.4 
let-7c 0.67 2.89 4.02 1.71 0.2 0.1 0.5 
let-7d 4.07 4.52 7.00 6.04 0.7 0.1 0.3 
let-7e 1.02 3.07 4.26 2.66 0.2 0.1 0.3 
let-7f 6.25 6.18 8.86 7.98 1.0 0.2 0.3 
let-7g −0.68 2.43 2.45 1.26 0.1 0.1 0.3 
let-7i 0.69 2.76 2.63 0.57 0.2 0.3 1.1 
miR-1 −3.19 4.35 6.83 7.26 0.0 0.0 0.0 
miR-7 4.65 6.57 8.22 6.07 0.3 0.1 0.4 
miR-9 5.53 6.13 10.07 6.19 0.7 0.0 0.6 
miR-10a 0.89 1.54 4.83 1.54 0.6 0.1 0.6 
miR-10b −0.69 1.38 4.00 0.09 0.2 0.0 0.6 
miR-15a 1.81 6.32 5.40 1.64 0.0 0.1 1.1 
miR-15b −0.41 0.13 3.51 1.27 0.7 0.1 0.3 
miR-16 −4.19 −2.77 0.36 −2.89 0.4 0.0 0.4 
miR-17 1.01 1.26 4.52 2.45 0.8 0.1 0.4 
miR-18a 4.40 2.02 7.05 4.37 5.2 0.2 1.0 
miR-18b 5.22 2.73 5.82 7.37 5.6 0.7 0.2 
miR-20a 0.44 1.21 4.02 1.66 0.6 0.1 0.4 
miR-20b 4.42 2.89 7.68 6.35 2.9 0.1 0.3 
miR-21 −4.43 −2.15 −1.71 −6.90 0.2 0.2 5.5 
miR-22 0.08 3.36 2.77 −0.17 0.1 0.2 1.2 
miR-23b −3.52 −3.00 1.21 −0.99 0.7 0.0 0.2 
miR-24 −2.70 −1.25 0.81 −1.73 0.4 0.1 0.5 
miR-26a −4.51 −2.56 −0.19 −2.40 0.3 0.1 0.2 
miR-33a 3.95 4.85 9.30 5.80 0.5 0.0 0.3 
miR-92a −2.66 −2.23 1.07 0.16 0.7 0.1 0.1 
miR-93 2.30 0.97 5.12 3.44 2.5 0.1 0.5 
miR-96 3.43 2.34 9.15 6.07 2.1 0.0 0.2 
miR-99a −2.32 −0.90 2.39 −1.22 0.4 0.0 0.5 
miR-100 −2.08 −0.42 2.35 −0.42 0.3 0.0 0.3 
miR-101 0.89 3.94 6.23 3.10 0.1 0.0 0.2 
miR-103 0.10 1.62 2.81 0.95 0.3 0.2 0.6 
miR-106b 2.51 2.79 5.90 3.34 0.8 0.1 0.6 
miR-122 8.10 7.33 3.40 2.23 1.7 26.0 58.7 
miR-124 6.65 1.50 9.02 9.98 35.5 0.2 0.1 
miR-125a-5p −2.24 −1.65 2.62 0.20 0.7 0.0 0.2 
miR-125b −5.55 −3.91 −0.32 −3.75 0.3 0.0 0.3 
miR-126 −3.38 −1.81 0.45 −1.96 0.3 0.1 0.4 
miR-127-5p 8.27 5.81 9.54 6.54 5.5 0.4 3.3 
miR-128a 1.31 9.16 5.92 3.44 0.0 0.0 0.2 
miR-129-5p 6.60 0.29 8.25 9.77 79.3 0.3 0.1 
miR-130a 2.67 5.24 7.29 5.46 0.2 0.0 0.1 
miR-132 3.25 0.97 6.70 3.80 4.9 0.1 0.7 
miR-133b 2.40 6.34 10.39 12.99 0.1 0.0 0.0 
miR-134 4.48 0.37 8.06 4.36 17.3 0.1 1.1 
miR-137 8.67 6.88 11.91 10.66 3.5 0.1 0.3 
miR-141 1.42 4.22 6.60 3.40 0.1 0.0 0.3 
miR-142-3p 2.60 5.07 3.43 3.06 0.2 0.6 0.7 
miR-142-5p 7.16 7.33 8.27 9.06 0.9 0.5 0.3 
miR-146a 0.18 0.74 3.44 3.14 0.7 0.1 0.1 
miR-146b-5p 0.80 1.26 3.98 1.62 0.7 0.1 0.6 
miR-150 −0.78 0.55 1.78 1.33 0.4 0.2 0.2 
miR-155 4.20 7.48 5.37 5.05 0.1 0.4 0.6 
miR-181a 2.42 1.94 4.68 2.06 1.4 0.2 1.3 
miR-182 3.39 2.99 7.81 7.42 1.3 0.0 0.1 
miR-183 4.28 7.92 8.70 7.62 0.1 0.0 0.1 
miR-185 2.34 3.78 5.58 3.68 0.4 0.1 0.4 
miR-192 3.78 3.81 6.16 4.40 1.0 0.2 0.7 
miR-194 3.76 3.92 6.50 4.50 0.9 0.1 0.6 
miR-195 −4.07 −2.89 0.79 −2.84 0.4 0.0 0.4 
miR-196a 1.66 2.81 5.97 2.42 0.5 0.1 0.6 
miR-205 −3.31 −1.59 0.39 −1.41 0.3 0.1 0.3 
miR-206 0.01 5.82 9.15 10.77 0.0 0.0 0.0 
miR-208 8.10 3.21 11.99 15.27 29.7 0.1 0.0 
miR-210 3.34 0.70 5.86 4.25 6.2 0.2 0.5 
miR-214 0.82 0.90 3.93 0.54 0.9 0.1 1.2 
miR-215 7.18 6.11 9.76 9.51 2.1 0.2 0.2 
miR-218 2.72 ND 7.14 4.72 — 0.0 0.3 
miR-219-5p 6.98 3.11 13.35 9.03 14.6 0.0 0.2 
miR-222 1.62 4.49 3.25 2.56 0.1 0.3 0.5 
miR-223 0.04 1.18 3.01 1.73 0.5 0.1 0.3 
miR-301a 6.59 4.97 9.89 7.72 3.1 0.1 0.5 
miR-302a 10.04 4.55 6.98 12.04 44.9 8.3 0.3 
miR-302c 12.91 6.50 10.40 11.85 85.0 5.7 2.1 
miR-345 5.83 1.96 7.12 6.16 14.6 0.4 0.8 
miR-370 7.54 3.97 5.78 6.59 11.9 3.4 1.9 
miR-371-3p 10.58 7.01 12.50 14.52 11.9 0.3 0.1 
miR-375 3.27 0.72 7.18 6.99 5.9 0.1 0.1 
miR-378 0.02 4.76 5.38 3.84 0.0 0.0 0.1 
miR-424 2.27 3.05 3.73 −2.21 0.6 0.4 22.2 
miR-452 4.57 4.77 9.19 7.02 0.9 0.0 0.2 
miR-488 11.36 4.95 12.78 12.77 85.0 0.4 0.4 
miR-498 13.41 8.30 17.00 19.47 34.5 0.1 0.0 
miR-503 7.17 3.78 9.40 6.45 10.5 0.2 1.7 
miR-518b 13.54 7.72 11.94 14.02 56.5 3.0 0.7 
miR-520g 7.57 6.76 10.58 9.36 1.8 0.1 0.3 
Normal ΔCtSSc ΔΔCtLSc ΔCtKeloid ΔΔCtSSc/Normal
Fold ChangeLSc/Normal
Fold ChangeKeloid/Normal
Fold Change
let-7a −2.94 0.24 0.67 −1.01 0.1 0.1 0.3 
let-7b −2.43 −0.07 0.06 −1.02 0.2 0.2 0.4 
let-7c 0.67 2.89 4.02 1.71 0.2 0.1 0.5 
let-7d 4.07 4.52 7.00 6.04 0.7 0.1 0.3 
let-7e 1.02 3.07 4.26 2.66 0.2 0.1 0.3 
let-7f 6.25 6.18 8.86 7.98 1.0 0.2 0.3 
let-7g −0.68 2.43 2.45 1.26 0.1 0.1 0.3 
let-7i 0.69 2.76 2.63 0.57 0.2 0.3 1.1 
miR-1 −3.19 4.35 6.83 7.26 0.0 0.0 0.0 
miR-7 4.65 6.57 8.22 6.07 0.3 0.1 0.4 
miR-9 5.53 6.13 10.07 6.19 0.7 0.0 0.6 
miR-10a 0.89 1.54 4.83 1.54 0.6 0.1 0.6 
miR-10b −0.69 1.38 4.00 0.09 0.2 0.0 0.6 
miR-15a 1.81 6.32 5.40 1.64 0.0 0.1 1.1 
miR-15b −0.41 0.13 3.51 1.27 0.7 0.1 0.3 
miR-16 −4.19 −2.77 0.36 −2.89 0.4 0.0 0.4 
miR-17 1.01 1.26 4.52 2.45 0.8 0.1 0.4 
miR-18a 4.40 2.02 7.05 4.37 5.2 0.2 1.0 
miR-18b 5.22 2.73 5.82 7.37 5.6 0.7 0.2 
miR-20a 0.44 1.21 4.02 1.66 0.6 0.1 0.4 
miR-20b 4.42 2.89 7.68 6.35 2.9 0.1 0.3 
miR-21 −4.43 −2.15 −1.71 −6.90 0.2 0.2 5.5 
miR-22 0.08 3.36 2.77 −0.17 0.1 0.2 1.2 
miR-23b −3.52 −3.00 1.21 −0.99 0.7 0.0 0.2 
miR-24 −2.70 −1.25 0.81 −1.73 0.4 0.1 0.5 
miR-26a −4.51 −2.56 −0.19 −2.40 0.3 0.1 0.2 
miR-33a 3.95 4.85 9.30 5.80 0.5 0.0 0.3 
miR-92a −2.66 −2.23 1.07 0.16 0.7 0.1 0.1 
miR-93 2.30 0.97 5.12 3.44 2.5 0.1 0.5 
miR-96 3.43 2.34 9.15 6.07 2.1 0.0 0.2 
miR-99a −2.32 −0.90 2.39 −1.22 0.4 0.0 0.5 
miR-100 −2.08 −0.42 2.35 −0.42 0.3 0.0 0.3 
miR-101 0.89 3.94 6.23 3.10 0.1 0.0 0.2 
miR-103 0.10 1.62 2.81 0.95 0.3 0.2 0.6 
miR-106b 2.51 2.79 5.90 3.34 0.8 0.1 0.6 
miR-122 8.10 7.33 3.40 2.23 1.7 26.0 58.7 
miR-124 6.65 1.50 9.02 9.98 35.5 0.2 0.1 
miR-125a-5p −2.24 −1.65 2.62 0.20 0.7 0.0 0.2 
miR-125b −5.55 −3.91 −0.32 −3.75 0.3 0.0 0.3 
miR-126 −3.38 −1.81 0.45 −1.96 0.3 0.1 0.4 
miR-127-5p 8.27 5.81 9.54 6.54 5.5 0.4 3.3 
miR-128a 1.31 9.16 5.92 3.44 0.0 0.0 0.2 
miR-129-5p 6.60 0.29 8.25 9.77 79.3 0.3 0.1 
miR-130a 2.67 5.24 7.29 5.46 0.2 0.0 0.1 
miR-132 3.25 0.97 6.70 3.80 4.9 0.1 0.7 
miR-133b 2.40 6.34 10.39 12.99 0.1 0.0 0.0 
miR-134 4.48 0.37 8.06 4.36 17.3 0.1 1.1 
miR-137 8.67 6.88 11.91 10.66 3.5 0.1 0.3 
miR-141 1.42 4.22 6.60 3.40 0.1 0.0 0.3 
miR-142-3p 2.60 5.07 3.43 3.06 0.2 0.6 0.7 
miR-142-5p 7.16 7.33 8.27 9.06 0.9 0.5 0.3 
miR-146a 0.18 0.74 3.44 3.14 0.7 0.1 0.1 
miR-146b-5p 0.80 1.26 3.98 1.62 0.7 0.1 0.6 
miR-150 −0.78 0.55 1.78 1.33 0.4 0.2 0.2 
miR-155 4.20 7.48 5.37 5.05 0.1 0.4 0.6 
miR-181a 2.42 1.94 4.68 2.06 1.4 0.2 1.3 
miR-182 3.39 2.99 7.81 7.42 1.3 0.0 0.1 
miR-183 4.28 7.92 8.70 7.62 0.1 0.0 0.1 
miR-185 2.34 3.78 5.58 3.68 0.4 0.1 0.4 
miR-192 3.78 3.81 6.16 4.40 1.0 0.2 0.7 
miR-194 3.76 3.92 6.50 4.50 0.9 0.1 0.6 
miR-195 −4.07 −2.89 0.79 −2.84 0.4 0.0 0.4 
miR-196a 1.66 2.81 5.97 2.42 0.5 0.1 0.6 
miR-205 −3.31 −1.59 0.39 −1.41 0.3 0.1 0.3 
miR-206 0.01 5.82 9.15 10.77 0.0 0.0 0.0 
miR-208 8.10 3.21 11.99 15.27 29.7 0.1 0.0 
miR-210 3.34 0.70 5.86 4.25 6.2 0.2 0.5 
miR-214 0.82 0.90 3.93 0.54 0.9 0.1 1.2 
miR-215 7.18 6.11 9.76 9.51 2.1 0.2 0.2 
miR-218 2.72 ND 7.14 4.72 — 0.0 0.3 
miR-219-5p 6.98 3.11 13.35 9.03 14.6 0.0 0.2 
miR-222 1.62 4.49 3.25 2.56 0.1 0.3 0.5 
miR-223 0.04 1.18 3.01 1.73 0.5 0.1 0.3 
miR-301a 6.59 4.97 9.89 7.72 3.1 0.1 0.5 
miR-302a 10.04 4.55 6.98 12.04 44.9 8.3 0.3 
miR-302c 12.91 6.50 10.40 11.85 85.0 5.7 2.1 
miR-345 5.83 1.96 7.12 6.16 14.6 0.4 0.8 
miR-370 7.54 3.97 5.78 6.59 11.9 3.4 1.9 
miR-371-3p 10.58 7.01 12.50 14.52 11.9 0.3 0.1 
miR-375 3.27 0.72 7.18 6.99 5.9 0.1 0.1 
miR-378 0.02 4.76 5.38 3.84 0.0 0.0 0.1 
miR-424 2.27 3.05 3.73 −2.21 0.6 0.4 22.2 
miR-452 4.57 4.77 9.19 7.02 0.9 0.0 0.2 
miR-488 11.36 4.95 12.78 12.77 85.0 0.4 0.4 
miR-498 13.41 8.30 17.00 19.47 34.5 0.1 0.0 
miR-503 7.17 3.78 9.40 6.45 10.5 0.2 1.7 
miR-518b 13.54 7.72 11.94 14.02 56.5 3.0 0.7 
miR-520g 7.57 6.76 10.58 9.36 1.8 0.1 0.3 

A mixture of equal amounts of miRNAs from three normal skins, three systemic scleroderma, three localized scleroderma, or three keloid were prepared, and miRNA expression profile in each disease in vivo was evaluated using PCR Array. The raw Ct was normalized using the values of small RNA housekeeping gene U6. ΔΔCt (the raw Ct of each miRNA − Ct of U6) were shown. The fold change was calculated as 1/2(ΔΔCt of each disease − ΔΔCt of normal skin).

We hypothesized that miRNAs overexpressed or suppressed both in SSc and LSc compared with normal skin, but not changed in keloid, may be important in the pathogenesis of SSc/LSc, because SSc/LSc and keloid are thought to have different etiology as described in Introduction. When a two-cycle difference (4-fold change in the ΔΔCT method) was considered meaningful, 10 miRNAs (let-7a, -7b, -7c, -7e, -7g, miR-10b, -15a, -22, -141, and -302c) matched the condition (Table I). Interestingly, 5 of the 10 miRNAs belonged to let-7 family, one of the first miRNA family discovered in human. Furthermore, although not more than a two-cycle difference, let-7d and -7i, remaining members of let-7 family, also tended to be decreased in SSc and LSc. Accordingly, we focused on let-7a, most downregulated let-7 member in both SSc and LSc: Compared with normal skin, the expression of let-7a was decreased in SSc skin (ΔΔCt −2.94 versus 0.24; 3.18-cycle difference = 0.11-fold change) and in LSc skin (ΔΔCt −2.94 versus 0.67; 3.61-cycle difference = 0.08-fold change).

We confirmed the array result by real-time PCR with specific primer for let-7a, using increased number of samples. The mean let-7a levels in seven SSc skin as well as seven LSc skin in vivo were significantly lower than those in seven normal skin or five keloid skin (Fig. 1A). The decrease of let-7a in SSc skin was milder than that in LSc skin, which is consistent with the array result.

FIGURE 1.

Expression levels of let-7a in SSc and LSc. (A) Mean relative transcript levels of let-7a expression in skin tissues of seven healthy controls (HC), seven SSc, seven LSc, and five keloid were determined by real-time PCR as described in 2Materials and Methods. The data are expressed as mean ± SD. The values in samples from HC were set at 1. *p < 0.05 by ANOVA. (B) In situ detection of let-7a in paraffin-embedded, formalin-fixed tissues of normal skin, SSc, and LSc (n = 3). The let-7a stained blue. Bar, 200 μm (upper panels) and = 50 μm (lower panels). (C) Mean relative levels of let-7a in cultured dermal fibroblasts derived from HC skin, LSc, and SSc. Data are expressed as the mean ± SD of three independent experiments. *p < 0.05. (D) let-7a levels in normal fibroblasts in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. Data are expressed as the mean ± SD of four independent experiments. *p < 0.05 compared with untreated normal fibroblasts (1.0).

FIGURE 1.

Expression levels of let-7a in SSc and LSc. (A) Mean relative transcript levels of let-7a expression in skin tissues of seven healthy controls (HC), seven SSc, seven LSc, and five keloid were determined by real-time PCR as described in 2Materials and Methods. The data are expressed as mean ± SD. The values in samples from HC were set at 1. *p < 0.05 by ANOVA. (B) In situ detection of let-7a in paraffin-embedded, formalin-fixed tissues of normal skin, SSc, and LSc (n = 3). The let-7a stained blue. Bar, 200 μm (upper panels) and = 50 μm (lower panels). (C) Mean relative levels of let-7a in cultured dermal fibroblasts derived from HC skin, LSc, and SSc. Data are expressed as the mean ± SD of three independent experiments. *p < 0.05. (D) let-7a levels in normal fibroblasts in the presence or absence of TGF-β1 (2 ng/ml) for 24 h. Data are expressed as the mean ± SD of four independent experiments. *p < 0.05 compared with untreated normal fibroblasts (1.0).

Close modal

In addition, in situ hybridization of skin tissue showed that signal for let-7a was evident in normal dermal fibroblasts but not in the fibroblasts of SSc and LSc (Fig. 1B). Moreover, relative let-7a levels in cultured dermal fibroblasts derived from SSc and LSc involved skin in vitro were also significantly lower than those in normal dermal fibroblasts (Fig. 1C). Thus, let-7a is thought to be decreased in dermal fibroblasts of SSc and LSc both in vivo and in vitro.

We tried to clarify the mechanism that mediates decreased expression of let-7a in SSc/LSc fibroblasts. To examine the possibility that the downregulation of let-7a is due to the stimulation of intrinsic TGF-β activation seen in these cell types as described in the 1Introduction, normal fibroblasts were stimulated with exogenous TGF-β1. TGF-β1 downregulated let-7a expression mildly but significantly (Fig. 1D), suggesting that downregulation of let-7a is consequence of activation of TGF-β signaling in SSc/LSc fibroblasts, at least partly.

Next, we determined the contribution of downregulated let-7a to the regulation of type I collagen in SSc and LSc fibroblasts. As described in previous reports (11, 36), the expression of both α1(I) and α2(I) chain, two major components of type I collagen, was significantly increased in SSc (Fig. 2A) and LSc (Fig. 2B) fibroblasts. In addition, both α1(I) and α2(I) collagen protein was induced by exogenous TGF-β1 in normal fibroblasts (Fig. 2C): the overexpression of type I collagen in SSc/LSc fibroblasts is thought to be due to intrinsic TGF-β1 activation (5, 11). Considering above results that let-7a expression is downregulated in SSc/LSc dermal fibroblasts as well as normal fibroblasts stimulated with exogenous TGF-β1 (Fig. 1), downregulation of let-7a may induce collagen expression.

FIGURE 2.

The effect of let-7a on the expression of type I collagen in human dermal fibroblasts. (A) Expression of type Ι collagen protein in cultured normal and SSc dermal fibroblasts were detected by immunoblotting. Type Ι collagen protein expression levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to the levels in normal fibroblasts (1.0). *p < 0.05 as compared with the values in normal fibroblasts. (B) Expression of type Ι collagen protein in cultured normal and LSc dermal fibroblasts are shown and quantitated as described in (A). *p < 0.05. (C) Type Ι collagen protein levels in normal fibroblasts treated with or without TGF-β1 (2 ng/ml) for 24 h is shown by immunoblotting. (D) Normal human fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control inhibitor or let-7a inhibitor for 96 h. Cell lysates were subjected to immunoblotting. Type Ι collagen protein expression levels were quantitated as described in (A) (n = 3). *p < 0.05 as compared with the value in cells with control inhibitor (1.0). (E) Fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with let-7a inhibitor or miScript Target protectors for the let-7a binding sites on α1(Ι) and α2(Ι) collagen for 96 h. Cell lysates were subjected to immunoblotting. (F) Normal human fibroblasts at a density of 1 × 104 cells/well in 96-well culture plates were transfected with luciferase reporters containing 3′-UTR segment of α1(I) collagen (□) or α2(I) collagen (▪) and the indicated miRNA inhibitors. The bar graph shows the relative luciferase activity; *p < 0.05 as compared with the values in cells with control inhibitor (1.0). (G) Normal human fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control miRNA precursor or let-7a precursor. Cell lysates were subjected to immunoblotting.

FIGURE 2.

The effect of let-7a on the expression of type I collagen in human dermal fibroblasts. (A) Expression of type Ι collagen protein in cultured normal and SSc dermal fibroblasts were detected by immunoblotting. Type Ι collagen protein expression levels quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to the levels in normal fibroblasts (1.0). *p < 0.05 as compared with the values in normal fibroblasts. (B) Expression of type Ι collagen protein in cultured normal and LSc dermal fibroblasts are shown and quantitated as described in (A). *p < 0.05. (C) Type Ι collagen protein levels in normal fibroblasts treated with or without TGF-β1 (2 ng/ml) for 24 h is shown by immunoblotting. (D) Normal human fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control inhibitor or let-7a inhibitor for 96 h. Cell lysates were subjected to immunoblotting. Type Ι collagen protein expression levels were quantitated as described in (A) (n = 3). *p < 0.05 as compared with the value in cells with control inhibitor (1.0). (E) Fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with let-7a inhibitor or miScript Target protectors for the let-7a binding sites on α1(Ι) and α2(Ι) collagen for 96 h. Cell lysates were subjected to immunoblotting. (F) Normal human fibroblasts at a density of 1 × 104 cells/well in 96-well culture plates were transfected with luciferase reporters containing 3′-UTR segment of α1(I) collagen (□) or α2(I) collagen (▪) and the indicated miRNA inhibitors. The bar graph shows the relative luciferase activity; *p < 0.05 as compared with the values in cells with control inhibitor (1.0). (G) Normal human fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control miRNA precursor or let-7a precursor. Cell lysates were subjected to immunoblotting.

Close modal

let-7a was the potent regulator of both α1(I) and α2(I) collagen, according to miRNA target gene predictions using the TargetScan (version 5.1, http://www.targetscan.org/), MiRanda (August 2010 release, http://www.microrna.org/), DIANAmicroT (version 3.0; http://diana.cslab.ece.ntua.gr/microT/), and PicTar (http://pictar.mdc-berlin.de/), leading programs in the field (3740). Actually, transfection of the specific inhibitor of let-7a in normal fibroblasts in vitro resulted in the statistically significant increase of protein expression of α1(I) and α2(I) collagen (Fig. 2D). In addition, to determine direct interaction between let-7a with collagen 3′-UTR, we used miRNA miScript Target Protectors (Qiagen), single-stranded, modified RNAs complementary to the let-7a binding sites on type I collagen 3′-UTR: the protectors cover the flanking regions of the binding sites and specifically interfere with the direct interaction between let-7a and collagen (http://www.qiagen.com/products/miscripttargetprotectors.aspx) (41). As shown in Fig. 2E, transfection of the let-7a protector resulted in the upregulation of type I collagen expression to the similar extent to let-7a inhibitor. Furthermore, we also performed luciferase reporter gene assay using luciferase constructs containing the α1(I) or α2(I) collagen 3′-UTR. let-7a inhibitor induced the luciferase activities compared with control inhibitor (Fig. 2F). In contrast, the transfection of let-7a precursor into normal fibroblasts downregulated protein expression of type I collagen (Fig. 2G). Taken together, our results indicated that α1(I) and α2(I) collagen is the direct target of let-7a and that let-7a plays some roles in the constitutively upregulated type I collagen expression in SSc/LSc fibroblasts.

Recently, many publications indicate serum miRNAs can be novel biomarkers in various diseases. Serum miRNAs are rather stable, because they are thought to be encapsulated in microvesicles and protected from RNase, extreme temperatures, extreme pHs, or freeze-thaw cycle (4246). Furthermore, miRNAs in the vesicles can be incorporated into other cells and may alter gene expression in the recipient cells (47). Thus, serum miRNA levels may not merely be secreted from apoptotic cells but may exert some biological effects. We evaluated the possibility that serum let-7a levels can be a disease marker of SSc/LSc.

There has been no report determining the expression of let-7a in cell-free body fluid. To confirm that let-7a was indeed present in human serum, miRNA was purified from human sera and let-7a was detected by quantitative real-time PCR using primer set specific for let-7a. As shown in Fig. 3A, the amplification of let-7a was observed, and Ct values were increased by the serial dilution of the miRNA. Thus, let-7a was detectable and quantitative in the serum using our method.

FIGURE 3.

Serum let-7a levels in patients with SSc and LSc. (A) The let-7a was present in serum sample. Serial dilution of cDNA (as is, 10-fold dilution, 100-fold dilation, and 0) synthesized from serum miRNA was used as template for real-time PCR. Amplification curves of gene-specific transcripts are shown to illustrate the process of exponential increase of fluorescence. (B) Serum let-7a concentrations measured by quantitative real-time PCR (normalized to cel-miR-54) in patients with DM, SLE, SSc, and LSc and in healthy control (HC) subjects are shown. let-7a concentrations are shown on the ordinate. The horizontal bar in each group shows the mean value. The minimum value in LSc patients was set at 1. M, morphea. *p < 0.05. (C) Correlations between clinical/serological features and serum levels of let-7a in patients with LSc. Correlations were assessed by Pearson’s correlation coefficient. Correlation coefficient (r2) and p values are shown.

FIGURE 3.

Serum let-7a levels in patients with SSc and LSc. (A) The let-7a was present in serum sample. Serial dilution of cDNA (as is, 10-fold dilution, 100-fold dilation, and 0) synthesized from serum miRNA was used as template for real-time PCR. Amplification curves of gene-specific transcripts are shown to illustrate the process of exponential increase of fluorescence. (B) Serum let-7a concentrations measured by quantitative real-time PCR (normalized to cel-miR-54) in patients with DM, SLE, SSc, and LSc and in healthy control (HC) subjects are shown. let-7a concentrations are shown on the ordinate. The horizontal bar in each group shows the mean value. The minimum value in LSc patients was set at 1. M, morphea. *p < 0.05. (C) Correlations between clinical/serological features and serum levels of let-7a in patients with LSc. Correlations were assessed by Pearson’s correlation coefficient. Correlation coefficient (r2) and p values are shown.

Close modal

Serum samples were obtained from 17 healthy control subjects, 39 SSc patients consisted of 20 dcSSc and 19 lcSSc, and 32 LSc patients consisted of 19 morphea, 8 LS, and 5 GM. We also included serum samples from other rheumatic diseases: eight patients with SLE and eight patients with DM. As shown in Fig. 3B, serum let-7a levels in LSc patients were significantly lower than those in healthy controls, which is consistent with the downregulation of let-7a in LSc skin. There was no significant difference in the values among the three groups of morphea, LS, and GM. In contrast, the decrease of let-7a levels in SSc sera was milder than that in LSc sera, which is consistent with the result in the skin. Although the mean serum let-7a level in dcSSc was decreased compared with that in lcSSc, we could not find statistical significance.

Next, we tried to clarify the correlation between clinical/serological features and serum levels of let-7a in patients with SSc. Patients with reduced serum let-7a (below the mean level of SSc patients) had significantly increased ratio of dcSSc:lcSSc, significantly higher modified Rodnan Total skin thickness score (modified Rodnan Total skin thickness score) and significantly lower frequency of anti-centromere Ab than those with elevated let-7a levels (Table II), suggesting that serum let-7a levels is inversely correlated with the severity of skin sclerosis. In contrast, correlations were assessed by Pearson’s correlation coefficient in four parameters in LSc patients: duration of disease (between symptom onset and the first visit to the hospital) reflects the subjective severity of symptom. The number of lesions (larger than 3 cm in diameter) correlates with the involved area. Soluble IL-2R is reported to be parallel with disease activity (27, 48). And ss-DNA is also a marker of disease activity, especially muscle involvement (49). As a result, there were no correlation (r < 0.4) between the serum let-7a levels and these four factors (Fig. 3C). Thus, decreased serum let-7a in LSc patients may not be associated with specific features of this disease.

Table II.
The correlation of the serum let-7a levels with the clinical and serological features in SSc patients
Clinical and Serological FeaturesPatients with Elevated let-7a Levels (n = 18)Patients with Reduced let-7a Levels (n = 21)p Value
Mean age at serum sampling, years 60.6 61.8 0.78 
Mean duration of disease, month 62.5 61.4 0.98 
dcSSc: lcSSc 5:13 15:6 0.01 
MRSS, score 7.4 14.9 0.04 
Clinical features    
 Pitting scars/ulcers 33.3 47.6 0.28 
 Nailfold bleeding 50.0 28.6 0.15 
 Raynaud’s phenomenon 83.3 76.2 0.44 
 Telangiectasia 27.8 28.6 0.62 
 Contracture of phalanges 72.2 71.4 0.62 
 Calcinosis 4.8 0.54 
Organ involvement    
 Pulmonary fibrosis 27.8 42.9 0.26 
 Esophagus 11.1 28.6 0.17 
 Heart 27.8 52.4 0.11 
 Kidney 9.5 0.28 
 Joints 27.8 9.5 0.14 
ANA specificity    
 Anti-topo I 16.7 33.3 0.21 
 Anti-centromere 66.7 28.6 0.02 
 Anti-U1 RNP 11.1 9.5 0.64 
Clinical and Serological FeaturesPatients with Elevated let-7a Levels (n = 18)Patients with Reduced let-7a Levels (n = 21)p Value
Mean age at serum sampling, years 60.6 61.8 0.78 
Mean duration of disease, month 62.5 61.4 0.98 
dcSSc: lcSSc 5:13 15:6 0.01 
MRSS, score 7.4 14.9 0.04 
Clinical features    
 Pitting scars/ulcers 33.3 47.6 0.28 
 Nailfold bleeding 50.0 28.6 0.15 
 Raynaud’s phenomenon 83.3 76.2 0.44 
 Telangiectasia 27.8 28.6 0.62 
 Contracture of phalanges 72.2 71.4 0.62 
 Calcinosis 4.8 0.54 
Organ involvement    
 Pulmonary fibrosis 27.8 42.9 0.26 
 Esophagus 11.1 28.6 0.17 
 Heart 27.8 52.4 0.11 
 Kidney 9.5 0.28 
 Joints 27.8 9.5 0.14 
ANA specificity    
 Anti-topo I 16.7 33.3 0.21 
 Anti-centromere 66.7 28.6 0.02 
 Anti-U1 RNP 11.1 9.5 0.64 

Unless indicated, the values are percentages.

ANA, Anti-nuclear Ab; Anti-topo I, anti-topoisomerase I Ab; Anti-centromere, anti-centromere Ab; MRSS, modified Rodnan total skin thickness score.

On the basis of the above results, we considered let-7 family, especially let-7a, as SSc/LSc-specific miRNA and a negative effector on fibrosis. Meanwhile, skin fibrosis induced by bleomycin injection in mice is known for a murine model of SSc/LSc (33). Indeed, as seen in SSc/LSc, the levels of let-7a in bleomycin-treated fibrotic mice skins were significantly lower than PBS-treated control mice skins (Fig. 4A). Then, we evaluated whether the supplementation of let-7a in vivo could prevent from bleomycin-induced skin fibrosis.

FIGURE 4.

The effect of let-7a on type Ι collagen expression in mice skin fibroblasts in vitro. (A) Bleomycin (100 μl) or PBS was injected intradermally into the shaved back of the 8-wk-old male BALB/cAJcl mice daily for 4 wk. let-7a levels in the back skin of bleomycin- or PBS-treated mice were determined by real-time PCR as described in 2Materials and Methods. Data are shown on the ordinate (n = 5). Bars show means. *p < 0.05. (BD) Cultured BALB/c mouse skin fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control small RNA or let-7a oligo (10 nM). (B) Relative levels of mature let-7a were analyzed by real-time PCR for the mature single-stranded form of let-7a. Data are shown on the ordinate (n = 4). Bars show means. The minimum value in controls was set at 1. *p < 0.05. (C) Type Ι collagen protein levels in the cell lysates were shown. (D) Relative mRNA levels of α1(Ι) and α2(Ι) collagen were analyzed by real-time PCR. The value in cells with control small RNA was set at 1. (E) Cultured mouse skin fibroblasts at a density of 1 × 104 cells/well in 96-well culture plates were transfected with luciferase reporters containing the 3′-UTR segment of α1(I) collagen (□) or α2(I) collagen (▪) and the indicated oligos. At 24 h after the transfection, luciferase activity was measured (n = 3). *p < 0.05 as compared with the value in cells with control small RNA (1.0). (F) Mean relative let-7a levels in mice back skin after let-7a injection into mice abdominal cavity. The line graphs represent; AteloGene and control small miRNA mixtures (Atelo + control; black), PBS and let-7a mixtures (PBS + let-7a; blue), and AteloGene and let-7a mixtures (Atelo + let-7a; red). Mice back skin was removed on days 1, 3, 5, and 7 after miRNA mixture injection. n = 4. *p < 0.05.

FIGURE 4.

The effect of let-7a on type Ι collagen expression in mice skin fibroblasts in vitro. (A) Bleomycin (100 μl) or PBS was injected intradermally into the shaved back of the 8-wk-old male BALB/cAJcl mice daily for 4 wk. let-7a levels in the back skin of bleomycin- or PBS-treated mice were determined by real-time PCR as described in 2Materials and Methods. Data are shown on the ordinate (n = 5). Bars show means. *p < 0.05. (BD) Cultured BALB/c mouse skin fibroblasts at a density of 2 × 104 cells/well in 24-well culture plates were transfected with control small RNA or let-7a oligo (10 nM). (B) Relative levels of mature let-7a were analyzed by real-time PCR for the mature single-stranded form of let-7a. Data are shown on the ordinate (n = 4). Bars show means. The minimum value in controls was set at 1. *p < 0.05. (C) Type Ι collagen protein levels in the cell lysates were shown. (D) Relative mRNA levels of α1(Ι) and α2(Ι) collagen were analyzed by real-time PCR. The value in cells with control small RNA was set at 1. (E) Cultured mouse skin fibroblasts at a density of 1 × 104 cells/well in 96-well culture plates were transfected with luciferase reporters containing the 3′-UTR segment of α1(I) collagen (□) or α2(I) collagen (▪) and the indicated oligos. At 24 h after the transfection, luciferase activity was measured (n = 3). *p < 0.05 as compared with the value in cells with control small RNA (1.0). (F) Mean relative let-7a levels in mice back skin after let-7a injection into mice abdominal cavity. The line graphs represent; AteloGene and control small miRNA mixtures (Atelo + control; black), PBS and let-7a mixtures (PBS + let-7a; blue), and AteloGene and let-7a mixtures (Atelo + let-7a; red). Mice back skin was removed on days 1, 3, 5, and 7 after miRNA mixture injection. n = 4. *p < 0.05.

Close modal

To our knowledge, there have been no reports on the technique for overexpressing miRNAs in the skin in vivo. We prepared let-7a oligo as described previously (31, 32) and before in vivo experiments, we evaluated whether it could reduce the collagen expression in vitro by transfecting into cultured mouse skin fibroblasts and measuring collagen RNA/protein levels and 3′-UTR luciferase activity.

The induction of let-7a in mouse dermal fibroblasts upregulated mature let-7a in cell lysates by the mature miRNA-specific PCR analysis (Fig. 4B) (26) and reduced type I collagen protein expression by immunoblotting (Fig. 4C). However, the mRNA expression of α1(I) or α2(I) collagen was not downregulated by let-7a induction (Fig. 4D), indicating that the let-7a oligo regulates the collagen translation without changing mRNA levels. In addition, the let-7a induction in mouse dermal fibroblasts reduced the luciferase activity of α1(I) or α2(I) collagen 3′-UTR compared with control small RNA (Fig. 4E). These results confirm that let-7a oligo could regulate the expression of collagen directly in mouse dermal fibroblasts.

Next, to deliver let-7a persistently into mice skin, control small RNA or let-7a oligo was mixed with atelocollagen (AteloGene) for the protection from degeneration by RNase in vivo (50). The mixtures were administered percutaneously into the abdominal cavity. Then we tried to confirm whether let-7a is overexpressed in the skin by our method; the let-7a levels in mice back skin were significantly increased by the injection of atelocollagen and let-7a mixture specifically compared with the mixture of atelocollagen and control small RNA (Fig. 4F). Such higher let-7a expressions continued until day 3 after injection. In contrast, the mixture of PBS and let-7a did not increase let-7a expression in the skin (Fig. 4F), probably because of the degradation.

Bleomycin was locally injected in the back of the BALB/c mice daily for 4 wk. At the same time, control small RNA or let-7a oligo mixed with atelocollagen was administered percutaneously into the abdominal cavity once per week (four times a month) (Fig. 5A). When PBS was injected in back skin instead of bleomycin, there was no difference in the skin between administration of control small RNA and let-7a (Fig. 5B). In contrast, in the presence of control small RNA, mice skin injected with bleomycin showed dermal fibrosis with increased number of thickened collagen bundles and strong Masson’s trichrome staining (Fig. 5C, left panel). Thus, the injection of control small RNAs did not have an antifibrotic effect (e.g., triggering an antiviral innate immune response that leads to anti-fibrotic signaling pathways). However, administration of let-7a decreased the dermal thickness and Masson’s trichrome staining in the dermis, (Fig. 5C, right panel), which suggested decreased collagen deposition. We confirmed that the improvement of bleomycin-induced dermal thickening by let-7a administration was statistically significant (Fig. 5D). For further investigation, we evaluated whether injection of let-7a oligo into the abdominal cavity could affect other internal organs. Bleomycin did not cause tissue fibrosis in the lung (Fig. 5E) or liver (Fig. 5F), and there were no apparent differences in H&E staining between mice with control small RNA and let-7a administration. Taken together, systemic administration of let-7a could lead to the let-7a overexpression in skin and the attenuation of skin fibrosis by bleomycin.

FIGURE 5.

systemic administration of let-7a in bleomycin-induced skin fibrosis in vivo. (A) The protocol for (B)–(F) is shown. Bleomycin was locally injected in the back of the BALB/c mice daily for 4 wk. At the same time, control small miRNA or let-7a mixed with atelocollagen were administered percutaneously into the abdominal cavity once per week (four times a month). The back skin was obtained 1 d after final bleomycin injection. (B) H&E staining of PBS-treated mice skin with control small miRNA (left panel) or let-7a (right panel) injection into the abdominal cavity. Scale bar, 100 μm. (C) H&E (upper panels) and Masson’s trichrome staining (lower panels) of bleomycin-treated mice skin with control miRNA (left panels) or let-7a (right panels) injection into the abdominal cavity. Scale bar, 100 μm. (D) Dermal thickness was measured in bleomycin-treated mice skin with control or let-7a injection into the abdominal cavity. Data are shown on the ordinate (n = 6). Bars show means. *p < 0.05. (E) H&E staining of the lung in PBS- or bleomycin-treated mice with injection of control miRNA (left panels) or let-7a (right panels) into the abdominal cavity. Scale bar, 100 μm. (F) H&E staining of the liver in PBS- or bleomycin-treated mice with injection of control miRNA (left panels) or let-7a (right panels) into the abdominal cavity. Scale bar, 100 μm.

FIGURE 5.

systemic administration of let-7a in bleomycin-induced skin fibrosis in vivo. (A) The protocol for (B)–(F) is shown. Bleomycin was locally injected in the back of the BALB/c mice daily for 4 wk. At the same time, control small miRNA or let-7a mixed with atelocollagen were administered percutaneously into the abdominal cavity once per week (four times a month). The back skin was obtained 1 d after final bleomycin injection. (B) H&E staining of PBS-treated mice skin with control small miRNA (left panel) or let-7a (right panel) injection into the abdominal cavity. Scale bar, 100 μm. (C) H&E (upper panels) and Masson’s trichrome staining (lower panels) of bleomycin-treated mice skin with control miRNA (left panels) or let-7a (right panels) injection into the abdominal cavity. Scale bar, 100 μm. (D) Dermal thickness was measured in bleomycin-treated mice skin with control or let-7a injection into the abdominal cavity. Data are shown on the ordinate (n = 6). Bars show means. *p < 0.05. (E) H&E staining of the lung in PBS- or bleomycin-treated mice with injection of control miRNA (left panels) or let-7a (right panels) into the abdominal cavity. Scale bar, 100 μm. (F) H&E staining of the liver in PBS- or bleomycin-treated mice with injection of control miRNA (left panels) or let-7a (right panels) into the abdominal cavity. Scale bar, 100 μm.

Close modal

miRNAs, short RNA molecules on average only 22 nucleotides long, bind to 3′-UTRs of target mRNAs and lead to gene silencing. Recent vigorous efforts of research in this field indicated that miRNAs play roles in the pathogenesis of various disorders (1618). This study demonstrated the role of let-7a in collagen overexpression and its contribution to the pathogenesis of fibrosis in SSc/LSc by several major findings.

First, we tried to identify miRNAs specifically up- or downregulated in SSc/LSc compared with normal or keloid skin using PCR array. Because the array analysis was performed as a single experiment, a statistical significance could not be evaluated. Therefore, we confirmed the result using real-time PCR, accompanied by statistical analysis: let-7a was significantly downregulated in SSc/LSc tissue. Our study is the first, to our knowledge, to demonstrate the let-7a downregulation in SSc/LSc. The downregulation of let-7a may result from activated endogenous TGF-β signaling. As the negative mediator of TGF-β signaling, let-7a may play a role in the constitutive up-regulation of type I collagen in these cells. However, because the effect of TGF-β stimulation on the let-7a expression in normal fibroblasts in this study was significant but mild, there may be other factors mediating the let-7a downregulation in SSc. Future studies should be needed to clarify this point.

Second, we found the new miRNA–mRNA target interactions: let-7 and type I collagen. miRNAs have been implicated in immune response as well as cell development, cell differentiation, proliferation, and apoptosis (51). Our study suggests miRNAs are also involved in the regulatory mechanisms of extracellular matrix metabolism.

Also, we first investigated serum miRNA levels in SSc/LSc and found let-7a can be a biomarker of SSc and LSc for the diagnosis or the evaluation of disease activity. Contrary to our expectation, let-7a levels in dermal fibroblasts and sera of LSc patients were lower than those in SSc. This may be explained by the notion that fibrosis in the lesion tends to be more severe in LSc compared with SSc; For example, the fibrosis in LSc sometimes extends to the muscular tissues or bone beneath the cutaneous lesions but not in SSc. Thus, lower let-7a levels may reflect severer tissue fibrosis in LSc. As described above, we considered let-7 family as the SSc/LSc-specific miRNAs. However, as seen in Table I, there were other miRNAs downregulated both in SSc and LSc, but not in keloid, including miR-10b, -15a, -22, -141, and -302c. The relation between these miRNAs and SSc/LSc is needed to be examined.

Last, we tried to determine the function of let-7a in vivo model. To our knowledge, this is the first report describing the method for miRNA overexpression in mice skin by i.p. miRNA injection, and our procedure may become a standard method for similar experiments in the future. The intermittent overexpression of let-7a by i.p. injection had therapeutic value against skin fibrosis induced by bleomycin. miRNA-targeted therapy for human diseases is expected to become a scientific breakthrough. Miravirsen (SPC3649), a specific inhibitor of miR-122, has finished Phase 2a clinical trials in patients infected with hepatitis C virus (52). Also, Nakasa et al. (53) have demonstrated that systemic injection of miR-146a prevents joint destruction in collagen-induced arthritic mice.

In summary, our hypothetical model is shown in Fig. 6. In normal fibroblasts, let-7a has a negative effect on type Ι collagen expression. However, downregulated let-7a by the stimulation of TGF-β contributes to the overexpression of type I collagen in SSc/LSc fibroblasts. Although there are thought to be so many factors regulating the fibrotic process in SSc and LSc, let-7a may also play some roles in the pathogenesis of these diseases. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approach against cutaneous fibrosis using miRNA.

FIGURE 6.

Schematic model of let-7a-induced collagen overexpression in SSc/LSc fibroblasts. In SSc/LSc fibroblasts, let-7a expression is constitutively decreased by TGF-β signaling, and subsequently collagen expression is induced, which results in tissue fibrosis seen in SSc/LSc.

FIGURE 6.

Schematic model of let-7a-induced collagen overexpression in SSc/LSc fibroblasts. In SSc/LSc fibroblasts, let-7a expression is constitutively decreased by TGF-β signaling, and subsequently collagen expression is induced, which results in tissue fibrosis seen in SSc/LSc.

Close modal

We thank Junko Suzuki and Chiemi Shiotsu for valuable technical assistance. The luciferase reporter plasmid containing α1(I) collagen 3′-UTR was provided by Dr. Norisato Mitsutake (Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan).

This work was supported in part by a grant for scientific research from the Japanese Ministry of Education, Science, Sports, and Culture, by project research on intractable diseases from the Japanese Ministry of Health, Labor, and Welfare, and by a research grant from Shiseido.

The sequences presented in this article have been submitted to the Gene Expression Omnibus microarray data repository (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE43469.

Abbreviations used in this article:

     
  • Ct

    threshold cycle

  •  
  • dcSSc

    diffuse cutaneous systemic scleroderma

  •  
  • DM

    dermatomyositis

  •  
  • GM

    generalized morphea

  •  
  • lcSSc

    limited cutaneous systemic scleroderma

  •  
  • LS

    linear scleroderma

  •  
  • LSc

    localized scleroderma

  •  
  • miRNA

    microRNA

  •  
  • SLE

    systemic lupus erythematosus

  •  
  • SSc

    systemic scleroderma

  •  
  • UTR

    untranslated region.

1
Fett
N.
,
Werth
V. P.
.
2011
.
Update on morphea: part I. Epidemiology, clinical presentation, and pathogenesis.
J. Am. Acad. Dermatol.
64
:
217
228, quiz 229–230
.
2
Fett
N.
,
Werth
V. P.
.
2011
.
Update on morphea: part II. Outcome measures and treatment.
J. Am. Acad. Dermatol.
64
:
231
242, quiz 243–244
.
3
Jablonska
S.
,
Rodnan
G.
.
1979
.
Localized forms of scleroderma.
Clin. Rheum. Dis.
5
:
215
241
.
4
Higley
H.
,
Persichitte
K.
,
Chu
S.
,
Waegell
W.
,
Vancheeswaran
R.
,
Black
C.
.
1994
.
Immunocytochemical localization and serologic detection of transforming growth factor β1: association with type I procollagen and inflammatory cell markers in diffuse and limited systemic sclerosis, morphea, and Raynaud’s phenomenon.
Arthritis Rheum.
37
:
278
288
.
5
Kubo
M.
,
Ihn
H.
,
Yamane
K.
,
Tamaki
K.
.
2001
.
Up-regulated expression of transforming growth factor β receptors in dermal fibroblasts in skin sections from patients with localized scleroderma.
Arthritis Rheum.
44
:
731
734
.
6
Asano
Y.
,
Ihn
H.
,
Yamane
K.
,
Jinnin
M.
,
Mimura
Y.
,
Tamaki
K.
.
2004
.
Phosphatidylinositol 3-kinase is involved in α2(I) collagen gene expression in normal and scleroderma fibroblasts.
J. Immunol.
172
:
7123
7135
.
7
Asano
Y.
,
Ihn
H.
,
Yamane
K.
,
Kubo
M.
,
Tamaki
K.
.
2004
.
Impaired Smad7‑Smurf-mediated negative regulation of TGF-β signaling in scleroderma fibroblasts.
J. Clin. Invest.
113
:
253
264
.
8
Asano
Y.
,
Ihn
H.
,
Yamane
K.
,
Jinnin
M.
,
Mimura
Y.
,
Tamaki
K.
.
2005
.
Differential effects of the immunosuppressant FK-506 on human α2(I) collagen gene expression and transforming growth factor β signaling in normal and scleroderma fibroblasts.
Arthritis Rheum.
52
:
1237
1247
.
9
Kahaleh
B.
1993
.
Immunologic aspects of scleroderma.
Curr. Opin. Rheumatol.
5
:
760
765
.
10
Takehara
K.
,
Sato
S.
.
2005
.
Localized scleroderma is an autoimmune disorder.
Rheumatology
44
:
274
279
.
11
Asano
Y.
,
Ihn
H.
,
Jinnin
M.
,
Mimura
Y.
,
Tamaki
K.
.
2006
.
Involvement of αvβ5 integrin in the establishment of autocrine TGF-β signaling in dermal fibroblasts derived from localized scleroderma.
J. Invest. Dermatol.
126
:
1761
1769
.
12
Ala-Kokko
L.
,
Rintala
A.
,
Savolainen
E. R.
.
1987
.
Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures.
J. Invest. Dermatol.
89
:
238
244
.
13
Smith
J. C.
,
Boone
B. E.
,
Opalenik
S. R.
,
Williams
S. M.
,
Russell
S. B.
.
2008
.
Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways.
J. Invest. Dermatol.
128
:
1298
1310
.
14
Fujiwara
M.
,
Muragaki
Y.
,
Ooshima
A.
.
2005
.
Keloid-derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration.
Br. J. Dermatol.
153
:
295
300
.
15
Syed
F.
,
Ahmadi
E.
,
Iqbal
S. A.
,
Singh
S.
,
McGrouther
D. A.
,
Bayat
A.
.
2011
.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
Br. J. Dermatol.
164
:
83
96
.
16
Davidson-Moncada
J.
,
Papavasiliou
F. N.
,
Tam
W.
.
2010
.
MicroRNAs of the immune system: roles in inflammation and cancer.
Ann. N. Y. Acad. Sci.
1183
:
183
194
.
17
Furer
V.
,
Greenberg
J. D.
,
Attur
M.
,
Abramson
S. B.
,
Pillinger
M. H.
.
2010
.
The role of microRNA in rheumatoid arthritis and other autoimmune diseases.
Clin. Immunol.
136
:
1
15
.
18
Herrera
B. M.
,
Lockstone
H. E.
,
Taylor
J. M.
,
Ria
M.
,
Barrett
A.
,
Collins
S.
,
Kaisaki
P.
,
Argoud
K.
,
Fernandez
C.
,
Travers
M. E.
, et al
.
2010
.
Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes.
Diabetologia
53
:
1099
1109
.
19
Friedman
R. C.
,
Farh
K. K.
,
Burge
C. B.
,
Bartel
D. P.
.
2009
.
Most mammalian mRNAs are conserved targets of microRNAs.
Genome Res.
19
:
92
105
.
20
LeRoy
E. C.
,
Black
C.
,
Fleischmajer
R.
,
Jablonska
S.
,
Krieg
T.
,
Medsger
T. A.
 Jr.
,
Rowell
N.
,
Wollheim
F.
.
1988
.
Scleroderma (systemic sclerosis): classification, subsets and pathogenesis.
J. Rheumatol.
15
:
202
205
.
21
Ihn
H.
,
Sato
S.
,
Fujimoto
M.
,
Kikuchi
K.
,
Igarashi
A.
,
Soma
Y.
,
Tamaki
K.
,
Takehara
K.
.
1996
.
Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis.
Clin. Exp. Immunol.
105
:
475
479
.
22
Sato
S.
,
Fujimoto
M.
,
Ihn
H.
,
Kikuchi
K.
,
Takehara
K.
.
1994
.
Clinical characteristics associated with antihistone antibodies in patients with localized scleroderma.
J. Am. Acad. Dermatol.
31
:
567
571
.
23
Falanga
V.
,
Medsger
T. A.
,
Reichlin
M.
.
1985
.
High titers of antibodies to single-stranded DNA in linear scleroderma.
Arch. Dermatol.
121
:
345
347
.
24
Ihn
H.
,
LeRoy
E. C.
,
Trojanowska
M.
.
1997
.
Oncostatin M stimulates transcription of the human α2(I) collagen gene via the Sp1/Sp3-binding site.
J. Biol. Chem.
272
:
24666
24672
.
25
Kroh
E. M.
,
Parkin
R. K.
,
Mitchell
P. S.
,
Tewari
M.
.
2010
.
Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).
Methods
50
:
298
301
.
26
Zhang
Y.
,
Guo
J.
,
Li
D.
,
Xiao
B.
,
Miao
Y.
,
Jiang
Z.
,
Zhuo
H.
.
2010
.
Down-regulation of miR-31 expression in gastric cancer tissues and its clinical significance.
Med. Oncol.
27
:
685
689
.
27
Ihn
H.
,
Sato
S.
,
Fujimoto
M.
,
Kikuchi
K.
,
Takehara
K.
.
1996
.
Clinical significance of serum levels of soluble interleukin-2 receptor in patients with localized scleroderma.
Br. J. Dermatol.
134
:
843
847
.
28
Ihn
H.
,
Yazawa
N.
,
Kubo
M.
,
Yamane
K.
,
Sato
S.
,
Fujimoto
M.
,
Kikuchi
K.
,
Soma
Y.
,
Tamaki
K.
.
2000
.
Circulating levels of soluble CD30 are increased in patients with localized scleroderma and correlated with serological and clinical features of the disease.
J. Rheumatol.
27
:
698
702
.
29
Martin
M. M.
,
Buckenberger
J. A.
,
Jiang
J.
,
Malana
G. E.
,
Nuovo
G. J.
,
Chotani
M.
,
Feldman
D. S.
,
Schmittgen
T. D.
,
Elton
T. S.
.
2007
.
The human angiotensin II type 1 receptor +1166 A/C polymorphism attenuates microrna-155 binding.
J. Biol. Chem.
282
:
24262
24269
.
30
Nuovo
G. J.
2008
.
In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations.
Methods
44
:
39
46
.
31
Kim
S. J.
,
Shin
J. Y.
,
Lee
K. D.
,
Bae
Y. K.
,
Sung
K. W.
,
Nam
S. J.
,
Chun
K. H.
.
2012
.
MicroRNA let-7a suppresses breast cancer cell migration and invasion through downregulation of C-C chemokine receptor type 7.
Breast Cancer Res.
14
:
R14
.
32
Yang
Q.
,
Jie
Z.
,
Cao
H.
,
Greenlee
A. R.
,
Yang
C.
,
Zou
F.
,
Jiang
Y.
.
2011
.
Low-level expression of let-7a in gastric cancer and its involvement in tumorigenesis by targeting RAB40C.
Carcinogenesis
32
:
713
722
.
33
Yamamoto
T.
,
Takagawa
S.
,
Katayama
I.
,
Yamazaki
K.
,
Hamazaki
Y.
,
Shinkai
H.
,
Nishioka
K.
.
1999
.
Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma.
J. Invest. Dermatol.
112
:
456
462
.
34
Tanaka
C.
,
Fujimoto
M.
,
Hamaguchi
Y.
,
Sato
S.
,
Takehara
K.
,
Hasegawa
M.
.
2010
.
Inducible costimulator ligand regulates bleomycin-induced lung and skin fibrosis in a mouse model independently of the inducible costimulator/inducible costimulator ligand pathway.
Arthritis Rheum.
62
:
1723
1732
.
35
Kashiyama
K.
,
Mitsutake
N.
,
Matsuse
M.
,
Ogi
T.
,
Saenko
V. A.
,
Ujifuku
K.
,
Utani
A.
,
Hirano
A.
,
Yamashita
S.
.
2012
.
miR-196a downregulation increases the expression of type I and III collagens in keloid fibroblasts.
J. Invest. Dermatol.
132
:
1597
1604
.
36
Mauch
C.
,
Kozlowska
E.
,
Eckes
B.
,
Krieg
T.
.
1992
.
Altered regulation of collagen metabolism in scleroderma fibroblasts grown within three-dimensional collagen gels.
Exp. Dermatol.
1
:
185
190
.
37
Lewis
B. P.
,
Burge
C. B.
,
Bartel
D. P.
.
2005
.
Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.
Cell
120
:
15
20
.
38
John
B.
,
Enright
A. J.
,
Aravin
A.
,
Tuschl
T.
,
Sander
C.
,
Marks
D. S.
.
2004
.
Human MicroRNA targets.
PLoS Biol.
2
:
e363
.
39
Kiriakidou
M.
,
Nelson
P. T.
,
Kouranov
A.
,
Fitziev
P.
,
Bouyioukos
C.
,
Mourelatos
Z.
,
Hatzigeorgiou
A.
.
2004
.
A combined computational-experimental approach predicts human microRNA targets.
Genes Dev.
18
:
1165
1178
.
40
Krek
A.
,
Grün
D.
,
Poy
M. N.
,
Wolf
R.
,
Rosenberg
L.
,
Epstein
E. J.
,
MacMenamin
P.
,
da Piedade
I.
,
Gunsalus
K. C.
,
Stoffel
M.
,
Rajewsky
N.
.
2005
.
Combinatorial microRNA target predictions.
Nat. Genet.
37
:
495
500
.
41
Long
J. M.
,
Lahiri
D. K.
.
2011
.
MicroRNA-101 downregulates Alzheimer’s amyloid-β precursor protein levels in human cell cultures and is differentially expressed.
Biochem. Biophys. Res. Commun.
404
:
889
895
.
42
Chen
X.
,
Ba
Y.
,
Ma
L.
,
Cai
X.
,
Yin
Y.
,
Wang
K.
,
Guo
J.
,
Zhang
Y.
,
Chen
J.
,
Guo
X.
, et al
.
2008
.
Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.
Cell Res.
18
:
997
1006
.
43
Mitchell
P. S.
,
Parkin
R. K.
,
Kroh
E. M.
,
Fritz
B. R.
,
Wyman
S. K.
,
Pogosova-Agadjanyan
E. L.
,
Peterson
A.
,
Noteboom
J.
,
O’Briant
K. C.
,
Allen
A.
, et al
.
2008
.
Circulating microRNAs as stable blood-based markers for cancer detection.
Proc. Natl. Acad. Sci. USA
105
:
10513
10518
.
44
Hunter
M. P.
,
Ismail
N.
,
Zhang
X.
,
Aguda
B. D.
,
Lee
E. J.
,
Yu
L.
,
Xiao
T.
,
Schafer
J.
,
Lee
M. L.
,
Schmittgen
T. D.
, et al
.
2008
.
Detection of microRNA expression in human peripheral blood microvesicles.
PLoS One
3
:
e3694
.
45
Valenti
R.
,
Huber
V.
,
Iero
M.
,
Filipazzi
P.
,
Parmiani
G.
,
Rivoltini
L.
.
2007
.
Tumor-released microvesicles as vehicles of immunosuppression.
Cancer Res.
67
:
2912
2915
.
46
Chen
X.
,
Liang
H.
,
Zhang
J.
,
Zen
K.
,
Zhang
C. Y.
.
2012
.
Horizontal transfer of microRNAs: molecular mechanisms and clinical applications.
Protein Cell
3
:
28
37
.
47
Valadi
H.
,
Ekström
K.
,
Bossios
A.
,
Sjöstrand
M.
,
Lee
J. J.
,
Lötvall
J. O.
.
2007
.
Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
Nat. Cell Biol.
9
:
654
659
.
48
Uziel
Y.
,
Krafchik
B. R.
,
Feldman
B.
,
Silverman
E. D.
,
Rubin
L. A.
,
Laxer
R. M.
.
1994
.
Serum levels of soluble interleukin-2 receptor. A marker of disease activity in localized scleroderma.
Arthritis Rheum.
37
:
898
901
.
49
Takehara
K.
,
Kikuchi
K.
,
Soma
Y.
,
Igarashi
A.
,
Ishibashi
Y.
.
1990
.
Anti-single-stranded DNA antibody and muscle involvement in localized scleroderma.
Arch. Dermatol.
126
:
1368
.
50
Takeshita
F.
,
Patrawala
L.
,
Osaki
M.
,
Takahashi
R. U.
,
Yamamoto
Y.
,
Kosaka
N.
,
Kawamata
M.
,
Kelnar
K.
,
Bader
A. G.
,
Brown
D.
,
Ochiya
T.
.
2010
.
Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes.
Mol. Ther.
18
:
181
187
.
51
Lu
T. X.
,
Hartner
J.
,
Lim
E. J.
,
Fabry
V.
,
Mingler
M. K.
,
Cole
E. T.
,
Orkin
S. H.
,
Aronow
B. J.
,
Rothenberg
M. E.
.
2011
.
MicroRNA-21 limits in vivo immune response-mediated activation of the IL-12/IFN-γ pathway, Th1 polarization, and the severity of delayed-type hypersensitivity.
J. Immunol.
187
:
3362
3373
.
52
Lanford
R. E.
,
Hildebrandt-Eriksen
E. S.
,
Petri
A.
,
Persson
R.
,
Lindow
M.
,
Munk
M. E.
,
Kauppinen
S.
,
Ørum
H.
.
2010
.
Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
Science
327
:
198
201
.
53
Nakasa
T.
,
Shibuya
H.
,
Nagata
Y.
,
Niimoto
T.
,
Ochi
M.
.
2011
.
The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis.
Arthritis Rheum.
63
:
1582
1590
.

The authors have no financial conflicts of interest.