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Keywords:

  • miR-181a;
  • TGF-β;
  • IL-6;
  • Mesenchymal stem cells;
  • MAPK

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Mesenchymal stem cells (MSCs) exhibit extensive self-renewal potential and can modulate immunocyte activation. Our previous study reported that miR-181a expression was significantly increased in placenta from women with severe preeclampsia (PE), but the mechanisms by which miR-181a regulates MSCs are unknown. In this study, we asked if and how miR-181a regulates MSCs' proliferation and immunosuppressive properties. We found that the expression of miR-181a in the MSCs derived from the umbilical cord and decidua of PE patients increased relative to MSCs derived from normal patients. Transfection with miR-181a oligos prevented MSCs proliferation but did not affect MSCs apoptosis. Overexpression of miR-181a blocked activation of the TGF-β signaling pathway and caused downregulation of target gene (TGFBR1 and TGFBRAP1) mRNA and protein expression. Reporter genes with putative miR-181a binding sites from the TGFBR1 and TGFBRAP1 3′-untranslated regions (3′-UTRs) were downregulated in the presence of miR-181a, suggesting that miR-181a binds to TGFBR1 and TGFBRAP1 3′-UTRs. In contrast, transfection of MSCs with miR-181a oligo enhanced expression of IL-6 and indoleamine 2,3-dioxygenase by activating p38 and JNK signaling pathways, respectively. MSCs transfected with miR-181a also enhanced the proliferation of T cells in a short-term culture. Additionally, treatment with control MSCs, but not miR-181a transfected MSCs, improved dextran sulfate sodium-induced experimental colitis, suggesting that miR-181a attenuates the immunosuppressive properties of MSCs in vivo. Together, our data demonstrate that miR-181a is an important endogenous regulator in the proliferation and immunosuppressive properties of MSCs. STEM Cells2012;30:1756–1770


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Mesenchymal stem cells (MSCs) have extensive self-renewal potential, can modulate immunocyte activation [1, 2], are easily expanded and stored ex vivo, and are considered to be “immunoprivileged cells.” Under stress conditions, MSCs decrease local inflammation and participate in tissue repair and regeneration by secreting cytokines, chemokines, and extracellular matrix proteins [3, 4]. Previous data from our group and others demonstrated the immunosuppressive and anti-inflammatory effects of MSCs in the treatment of several animal disease models, including autoimmune diseases [5–7]. As a result of these unique qualities, MSCs are attractive candidates in stem cell-based strategies for tissue repair.

Preeclampsia (PE) is a condition characterized by hypertension and proteinuria during pregnancy (after 20 weeks of gestation). Its pathogenesis is not completely understood. Previous studies indicate that PE may be a pregnancy-induced autoimmune disease, and that the unbalanced immune system in the maternal–fetal interface may be one cause of PE [8–10]. Maternal–fetal interface is an important source of MSCs, besides, several groups have isolated MSCs from umbilical cord, placenta, and decidua [11–13]. Aberrant levels of cytokines were observed in placenta-derived MSCs from PE patients, and higher levels of MSC negative markers were found in the placentas from PE patients [14, 15]. These findings suggested that MSCs could contribute to PE pathogenesis. In this study, we showed that systemic infusion with normal MSCs has beneficial effects on healing in a mouse model of PE induced by adoptive transfer of activated Th1 cells. In addition, we provided data on the regulatory role of microRNAs (miRNAs or miRs) in self-renewal and immunosuppressive of MSCs at the maternal–fetal interface.

miRNAs represent new dimension of gene regulation discovered in recent years. They are short (19–25 nucleotides) single-stranded non-protein-coding RNAs [16] that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs [17]. They function in diverse biological processes, including development, differentiation, apoptosis, and oncogenesis. We previously reported that miR-181a expression was significantly increased in placental samples from women with severe PE [18]. This result was confirmed in two subsequent reports [19, 20]. miR-181a is strongly expressed in bone marrow (BM) and thymus, can modulate hematopoietic lineage differentiation, and has an important role during T- and B-cell development [21–23]. miR-181a acts as an intrinsic antigen sensitivity “rheostat” in T cells. Abnormally low miR-181a expression was reported in patients with acute myeloid leukemia, chronic lymphocytic leukemia, non-small cell lung cancer, adult T-cell leukemia, and glioblastoma [24–28]. Additionally, miR-181a displays tumor suppressive effects against oral squamous cell carcinoma and glioma cells by suppressing cell proliferation [29, 30]. These findings indicate that miR-181a can suppress proliferation of cancer cells and regulate immunocyte activation. However, the exact function of miR-181a in MSCs has not been well-characterized.

In this study, we analyzed the expression of miR-181a in MSCs and found an increased expression of miR-181a in MSCs from PE patients, as compared to MSCs from normal patients. We also found that expression of TGFBR1 and TGFBRAP1, two master TGF-β signaling pathway regulators, was regulated by miR-181a. Our gain- or loss-of-function experiments demonstrated that miR-181a acts as an inhibitor of TGF-β signaling pathway in MSCs and prevents the proliferation of MSCs. miR-181a also enhanced expression of IL-6, VEGF, and indoleamine 2,3-dioxygenase (IDO) by activating the mitogen-activated protein kinases (MAPK) pathway and attenuated MSCs immunosuppressive properties in vitro and vivo. Thus, our data suggest that miR-181a activity underlies PE, and that aberrant upregulation of miR-181a leads to downregulation of TGF-β signaling and upregulation of MAPK signaling in PE patients. These findings will aid in our understanding of miRNA function in MSCs and may provide a basis for developing potential novel therapies for PE.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Culture of Human MSCs

Human umbilical cords and deciduas were obtained from the Department of Gynecology and Obstetrics, the Affiliated Drum Tower Hospital of Nanjing University Medical School from full-term caesarian section births. Umbilical arteries and vein were removed, and the remaining tissue was transferred to a sterile container in Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Grand Island, NY, http://www.invitrogen.com) with antibiotics (penicillin 100 μg/ml, streptomycin 10 μg/ml; Invitrogen Life Sciences) and was diced into 1–2 mm3 fragments. The tissue was incubated in an enzyme cocktail (hyaluronidase 5 U/ml, collagenase 125 U/ml, and dispase 50 U/ml; SIGMA) for 45–60 minutes with gentle agitation at 37°C. This tissue was then crushed with forceps to release individual cells and large pieces of tissue were removed. The cells were pelleted by low-speed centrifugation (250g for 5 minutes), suspended in fresh medium, and transferred to six-well plates containing DMEM/F12 supplemented with 20% fetal bovine serum. Cells were incubated at 37°C in an incubator with 5% CO2 at saturating humidity. When cells reached 70%–80% confluence or when numerous colonies were observed, the cells were detached with 0.25% trypsin-EDTA (Invitrogen, Carlsbad, CA, http://www.invitrogen.com); the trypsin was inactivated with fresh media. MSCs were plated at a density of 1 × 105 cells per milliliter in culture flasks (25 cm2) and maintained at 37°C in a humidified atmosphere containing 5% CO2 and were passaged when cells reached 70%–80% confluence. After four cell passages, the adherent cells showed surface antigens CD105+CD73+CD90+HLA-ABC+CD29+CD44+ and were lacking of surface antigens CD106−HLA-DR−CD19−CD11b−CD14−CD34−CD31−.

Quantitative Reverse Transcription Polymerase Chain Reaction Analysis

Total RNA was extracted using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. RNA integrity was determined using formaldehyde denaturalization agarose gel electrophoresis. RNA concentrations were measured with the SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA, http://www.bio-rad.com). The method to quantify mature miRNA was performed as described previously [18]. Primer oligonucleotides were synthesized by Invitrogen and are listed in Table 1.

Table 1. MicroRNA and mRNA primer information
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Western Blotting and ELISA

The method of Western blotting was performed as described previously [31]. Recombinant human TGF-β was from Peprotech (Rocky Hill, NJ, http://www.peprotech.com), MAPK inhibitor SB203580 was from Alexis Biochemicals Inc. (San Diego, CA, http://www.axxora.com), and SP600125 and PD98059 were from Beyotime (Shanghai, People's Republic of China, http://www.beyotime.com). The type I TGF-β receptor (TβRI) inhibitor SB431542, STAT1 inhibitor, and nuclear factor-κB (NF-κB) inhibitor pyrrolidinedithiocarbamate (PDTC) were from Alexis Biochemicals Inc. The anti-human c-Jun NH2-terminal kinase (JNK), phos-JNK, p38, phos-p38, extra cellular regulated protein kinases (ERK), phos-ERK, and β-tubulin antibodies for Western blot were from Cell Signaling Technology Inc. (Danvers, MA, http://www.cellsignal.com). The antibodies against human TGFBR1, IDO, and TGFBRAP1 for Western blot were from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com).

Protein levels of IL-6 and VEGF were measured in cell supernatants, by enzyme-linked immunosorbent assay (ELISA). Human precoated ELISA kits were used according to the instructions of the manufacturer (DAKEWE, Beijing, China, http://www.dakewe.net).

Plasmids Construction

Human genomic DNA served as template to amplify TGFBR1-3′-UTR-1, TGFBR1-3′-UTR-2, and TGFBRAP1-3′-UTR. The amplified polymerase chain reaction (PCR) products were gel-purified and digested with XbaI (Takara, Otsu, Japan, http://www.takara.co.jp). They were inserted into XbaI sites of the PGL3 vector (Promega, Madison, WI, http://www.promega.com), resulting in PGL3-TGFBR1-3UTR-1, PGL3-TGFBR1-3UTR-2, and PGL3-TGFBRAP1-3UTR.

Transient Transfection and Reporter Assay

Mimic endogenous precursor miRNAs, hsa-miR-181A-1 (pre-miR-181a), miRNA control #1 (pre-nc), anti-miR miRNA inhibitors, anti-miR-181a, and anti-miR negative control #1 (anti-nc) were obtained from Ambion (Austin, TX, http://www.ambion.com). Transfection of MSCs and HEK293A cells with miRNAs was done using Lipofectamine 2000 (Invitrogen). HEK293A cells were cotransfected with the luciferase reporter constructs described above (200 ng), pRL-CMV (20 ng; Promega), and the appropriate miRNA precursor. After 36 hours, cells were washed and lysed with passive lysis buffer (Promega), and f-luc and Renilla luciferase (r-luc) activities were determined using the dual-luciferase reporter assay system (Promega). The relative reporter activity was obtained by normalization to the r-luc activity.

Flow Cytometry

The following murine mAbs (from BD Biosciences, San Diego, http://www.bdbiosciences.com), purified or directly conjugated with fluorescein isothiocyanate (FITC), PE, or allophycocyanin (APC), were used in fluorescence activated cell sorting (FACS) analysis: anti-CD105, anti-CD73, anti-CD90, anti-HLA-ABC (major histocompatibility complex class I), anti-CD29, anti-CD44, anti-CD106, anti-HLA-DR (major histocompatibility complex class II), anti-CD19, anti-CD11b, anti-CD14, anti-CD34, anti-CD31, anti-CD45, and IgG and IgM isotype controls. For fluorescence measurements only, data from 10,000 single cell events were collected using a standard FACScalibur flow cytometer (Immunocytometry Systems; Becton, Dickinson, Franklin Lakes, NJ, http://www.bd.com). Data were analyzed using CELLQuest (Becton Dickinson) or FlowJo software (Treestar, Inc., San Carlos, CA).

Cell Viability Analysis

Cell viability was assessed with a Cell Counting Kit (Dojindo Laboratories Inc., Japan), as described previously [31].

Mixed Lymphocyte Reaction

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult donors after obtaining written, informed consent. PBMCs were isolated from heparinized venous blood by Ficoll-Paque gradient centrifugation. T cells were purified by anti-CD3 mAb-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer's instructions. Purified T cells were seeded in 96-well flat-bottomed plates with MSCs (MSCs:T cells = 1:5, 1:10, 1:20, and 1:50; 105 total cells) in a final volume of 200 μl 1640 medium. Phytohaemagglutinin (PHA; 5 μg/ml; Sigma) was used to induce proliferation of T cells, and the [3H] incorporation assay was used to quantify T-cell proliferation. The IDO inhibitor was from Sigma, and the IL-6 neutralizing Ab was from eBioscience (San Diego, CA).

Experimental Colitis Models and Evaluation of Severity of Colitis

Experimental colitis was induced by giving drinking water containing 3% dextran sulfate sodium (DSS) ad libitum for 8 days. The DSS solution was replaced with water on day 8, and mice continued to drink regular water daily until they were evaluated on day 13. C57BL/6 mice were randomly divided into the following groups: (a) naïve group without any treatment (n = 11), (b) DSS-treated group (n = 11), (c) DSS with human MSCs treatment (n = 11), and (d) DSS with pre-181a transfected MSCs treatment (n = 11). Control and pre-181a transfected MSCs (106) were resuspended in 100 μl of phosphate buffer saline (PBS) and were injected (i.p.) into mice 1 and 3 days after initiation of DSS treatment. Colitis severity was scored (0–4) by daily monitoring of weight loss, stool consistency/diarrhea, and presence of fecal bleeding. Mice were sacrificed by CO2 euthanasia, and the entire colon was quickly removed and gently cleared of feces with sterile PBS. We evaluated colon length and weight. Colon length was defined as the length from the rectum to the jejunum. For mRNA and protein extraction, colon segments were rapidly frozen in liquid nitrogen. For histopathological analysis, colon segments were fixed in 10% buffered formalin phosphate and paraffin-embedded sections were prepared for H&E staining. Histological scores were blindly determined as previously described [32].

Mouse Model for PE

The method of PE-like mouse model was performed as described previously [33].

Statistical Analysis

Results were presented as mean ± SEM. Statistical significance between groups was analyzed by one-way ANOVA followed by the Student–Newman–Keuls multiple comparisons tests. A p value of <.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Increased Expression of miR-181a in PE MSCs Prevents MSCs' Proliferation Through TGF-β Signaling

We isolated MSCs from umbilical cord (UCMSCs) and decidua from women with severe late-onset PE who delivered after 34 weeks and women with normal pregnancy. We then analyzed expression of miR-181a in these two MSC populations. The levels of miR-181a were 1.5-fold and 1.9-fold higher in PE MSCs derived from decidua (Fig. 1A) and umbilical cord than in normal MSCs derived from decidua and umbilical cord tissues (Fig. 1B), respectively. Because MSCs derived from umbilical cord tissues were easier to obtain than decidua, MSCs for the following experiments were all derived from umbilical cord tissue. To understand the functional consequences of elevated miR-181a expression in PE MSCs, we transfected a miR-181a-specific precursor, pre-miR-181a (pre-181a), or inhibitor, anti-miR-181a (anti-181a), for gain- or loss-of-function experiments, respectively. Transfection of MSCs with pre-miR-181a caused significant upregulation of miR-181a expression, while transfection with anti-miR-181a caused significant downregulation of miR-181a expression, as compared to controls (Fig. 1C, 1D). Normal and miR-181a transfected MSCs maintained similar cell morphology (Fig. 1E) (i.e., elongated and spindled shaped) and phenotypic surface antigens of MSCs (Supporting Information Fig. 1). We noted that PE-derived MSCs divided less frequently than MSCs derived from normal patients (Supporting Information Fig. 2). A cell counting kit was used to determine whether the change of miR-181a expression affects MSCs' proliferation (Fig. 1F). We found that elevated miR-181a expression in MSCs significantly decreased proliferation, as compared with control cells, 48 hours after transfection. Proliferation of MSCs transfected with anti-miR-181a was not significantly different than that of control MSCs. By contrast, neither elevation nor reduction of miR-181a expression affected MSCs apoptosis (Supporting Information Fig. 3). These results indicated that miR-181a inhibits proliferation but does not affect apoptosis. To understand how miR-181a inhibits MSCs proliferation, we used Targetscan (http://www.targetscan.org) and probability of interaction by target accessibility (PITA) (http://genie.weizmann.ac.il/pubs/mir07/mir07_predic tion.html) to identify candidate miR-181a target genes. In addition, CapitalBio Molecule Annotation System V3.0 was used to perform pathway analysis on the putative miR-181a target genes (http://bioinfo.capitalbio.com/mas3/). We found that several genes encoding members of the TGF-β signaling pathway were potential targets of miR-181a (Table 2). We pretreated MSCs with an inhibitor of the TβRI kinase SB431542 for 30 minutes before applying TGF-β1 treatment for another hour. SB431542 blocked the phosphorylation of Smad2 (Supporting Information Fig. 4A), and inhibited the proliferation of MSCs, even in the absence of TGF-β treatment (Supporting Information Fig. 4B). Next, MSCs were transfected with pre-miR-181a for 48 hours before being treated with TGF-β for 1 hour. Under these conditions, we also found that cell proliferation decreased and phosphorylation of Smad2 was blocked (Fig. 1G, 1H). Additionally, PAI-1 expression, a classic TGF-β1 target gene regulated by Smad signaling, was also affected by pre-miR-181a transfection (Supporting Information Fig. 5). These results suggested that pre-miR-181a influenced MSCs proliferation via TGF-β signaling.

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Figure 1. Increased expression of miR-181a in PE MSCs prevent proliferation of MSCs through TGF-β signaling. The passage 1 MSCs derived from deciduas or umbilical cord tissues were used to detect the basal expression of miR-181a. (A): Basal expression of miR-181a in MSCs from normal deciduas (n = 16) and from patients with PE deciduas (n = 13). Bars show the mean. p values were determined by Mann–Whitney U test with Bonferroni correction. p = .0154. (B): Basal expression of miR-181A in MSCs from normal umbilical cord tissues (n = 16) and from patients with PE umbilical cord tissues (n = 11). Bars show the mean. p values were determined by Mann–Whitney U test with Bonferroni correction. p = .0167. MSCs were transfected with control (pre-nc or anti-nc), pre-miR-181a, and anti-miR-181a individually. 30 pmol of pre-miR-181a or anti-miR-181a was used for transfection of 8 × 104 cells in 12-well plates. After 48 hours of transfection, MSCs were collected and analyzed. (C, D): Quantitative reverse transcription polymerase chain reaction analysis of the expression of miR-181a in the MSCs 48 hours after transfection (n = 3). (E): Morphology of MSCs 48 hours after transfection (×20). (F): MSCs proliferation determined by Cell Counting Kit 48 hours after transfection (n = 4). (G): Phosphorylation of Smad2 after pre-181a transfection. (H): MSCs proliferation determined by cell counting kit after pre-181a transfection and treated with TGF-β1 (n = 4). All data are representative of three independent experiments. Abbreviations: DMSC, MSCs from deciduas; miRNA, microRNA; MSCs, mesenchymal stem cells; PE, preeclampsia; UCMSC, MSCs from umbilical cord tissues.

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Table 2. Several genes of TGF-β signaling pathway were potential target genes of miR-181a, predicted with PITA
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miR-181a Regulates TGF-β Signaling Through TGFBR1 and TGFBRAP1

To investigate the specific TGF-β pathway genes targeted by miR-181a, we used quantitative reverse transcription PCR (qRT-PCR) to determine expression level of potential miR-181a target genes in the transfected MSCs. miR-181a expression caused significant downregulation of TGFBR1 and TGFBRAP1 mRNA levels (Fig. 2A). Western blot analysis showed that the protein levels of TGFBR1 and TGFBRAP1 were also downregulated (Fig. 2B). Expression of TGFBR1 and TGFBRAP1 mRNA and protein was also analyzed in MSCs transfected with anti-miR-181a. Consistently, we found that TGFBR1 and TGFBRAP1 were upregulated when miR-181a activity was compromised (Supporting Information Fig. 6A, 6B).

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Figure 2. miR-181a regulates TGF-β signaling pathway members TGFBR1 and TGFBRAP1. (A): Quantitative reverse transcription polymerase chain reaction analysis of the expression of predicted target genes 48 hours after pre-181a transfection (n = 3). (B): Analysis of the expression of miR-181a target genes TGFBR1 and TGFBRAP1 by Western blot 48 hours after pre-181a transfection. (C): Schematic description of conserved binding sites for miR-181a. The seed-recognizing sites were demarked and all nucleotides in these regions were completely conserved in several species. Hypothesized duplexes between miR-181a and the binding sites in TGFBR1 and TGFBRAP1 3′-UTRs are illustrated. The predicted free energy of each hybrid is also indicated. (D): Schematic representation of reporter plasmids PGL3-TGFBR1-3UTR-1, PGL3-TGFBR1-3UTR-2, and PGL3-TGFBRAP1-3UTR. Two parts of the TGFBR1-3′-UTR was subcloned downstream of the f-luc open reading frame, each contains one of the putative miR-181a pairing sites. The complete TGFBRAP1-3UTR was also cloned into PGL3 vector. (E): Direct recognition of TGFBR1 and TGFBRAP1 3′-UTR by miR-181a. For luciferase reporter assays in 24-well plates, 200 ng luciferase reporter plasmid, 20 ng pRL-CMV (transfection control; Ambion) and indicated amounts (15, 30, and 60 pmol) of pre-181a were transfected. After 36 hours of transfection, cells were assayed using luciferase assay kits (Promega) (n = 3). (F): Pearson's correlation scatter plot of the expression of miR-181a and TGFBR1 mRNA from normal umbilical cord tissues (n = 9) and from patients with PE umbilical cord tissues (n = 7). (G): Pearson's correlation scatter plot of the expression of miR-181a and TGFBRAP1 mRNA from normal umbilical cord tissues (n = 9) and from patients with PE umbilical cord tissue (n = 7). Data are representative of three independent experiments. Abbreviations: CDS, coding sequence; PE, preeclampsia; UTR, untranslated region.

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An on-line search of the Targetscan database demonstrated that two potential miR-181a binding sites are found in the TGFBR1 3′-UTR. The free energy values of hybrids that would be generated by miRNA binding are −19.6 and −11.6 kcal/mol. Also, two miR-181a binding sites in the TGFBRAP1 3′-UTR were observed. The free energy values of the hybrids formed at these sites would be −24.5 and −19.1 kcal/mol (Fig. 2C).

To experimentally validate the computational data, two parts of the TGFBR1-3′-UTR, each containing a putative miR-181a binding site, were subcloned downstream of the f-luc open reading frame in the PGL3 vector (Fig. 2D). The complete TGFBRAP1-3′-UTR was subcloned similarly. These reporter constructs (PGL3-TGFBR1-3UTR-1, PGL3-TGFBR1-3UTR-2, and PGL3-TGFBRAP1-3UTR) were each cotransfected in the HEK293A cell line with pRL-CMV (to normalize for transfection differences) and either miR-control (pre-nc) or pre-miR-181a at varying concentrations.

The relative luciferase activities were all markedly diminished in cells cotransfected with the three reporter constructs and miR-181a. Overexpression of miR-181a resulted in a 30% reduction of firefly luciferase reporter activity in cells cotransfected with the PGL3-TGFBR1-3UTR-1 or PGL3-TGFBR1-3UTR-2 and a 60% reduction in cells cotransfected with PGL3-TGFBRAP1-3UTR (Fig. 2E). These results indicate that miR-181a can interfere with mRNA translation via direct interaction with the TGFBR1-3UTR and TGFBRAP1-3UTR.

Consistent with these findings, expression of TGFBR1 and TGFBRAP1 mRNA in MSCs derived from PE patients' umbilical cord tissues were both strongly inversely correlated with basal miR-181a levels. By contrast, the expression of TGFBR1 and TGFBRAP1 mRNA in MSCs derived from normal term pregnancy umbilical cord tissues were weakly or not correlated with basal miR-181a levels (Fig. 2F, 2G).

miR-181a Enhances Expression of IL-6, VEGF, and IDO in MSCs

We next examined the effect of miR-181a on the immunosuppressive properties of MSCs. MSCs secrete several soluble factors to suppress immune responses. To determine whether overexpression of miR-181a has any affect on the expression of these secreted soluble factors, we analyzed their expression by qRT-PCR. We measured mRNA for IL-6, TGFB, IDO, VEGF, and COX2 in pre-miR-181a and pre-miR-control transfected MSCs. After 48 hours, expression of IDO, IL-6, and VEGF mRNAs were 10-fold, fivefold, and fourfold higher in pre-miR-181a transfected MSCs than control MSCs, respectively (Fig. 3A). Expression of TGFB and COX2 transcripts were not significantly influenced by pre-miR-181a transfection. MSCs transfected with pre-miR-181a also produced significantly more IL-6 and VEGFA protein than pre-nc transfected MSCs (Fig. 3B, 3C). However, IDO protein could not be detected by Western blot or ELISA in MSCs transfected with pre-miR-181a or pre-nc (data no show). Transfection of MSCs with anti-miR-181a caused a modest decrease in IL-6 mRNA and protein expression and IDO mRNA expression (Supporting Information Fig. 7A, 7B), as compared with anti-nc transfected MSCs.

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Figure 3. miR-181a enhances IL-6, VEGF, and IDO expression in mesenchymal stem cells (MSCs). MSCs were transfected with control (pre-nc and anti-nc), pre-miR-181a, and anti-miR-181a individually. (A): Quantitative reverse transcription polymerase chain reaction analysis of the expression of IL-6, TGFβ, IDO, VEGF, and COX2 48 hours after transfection (n = 3). The protein level of IL-6 (B) and VEGF-A (C) expressions were detected by ELISA 48 hours after pre-181a transfection (n = 3). (D): Pre-181a transfected MSCs, pre-nc transfected MSCs, and control MSCs were treated with indicated concentrations (0, 10, 50, 100, 200, 500, 1,000, 1,500, and 2,000 U/ml) of IFN-γ for 24 hours and then cells were harvested and total RNA was extracted. Quantitative mRNA expression was measured as described before (n = 3). (E): Pearson's correlation scatter plot of the expression of miR-181a and IL-6 mRNA in MSCs derived from normal umbilical cord tissues (n = 9) and from patients with PE umbilical cord tissues (n = 7). (F): Pearson's correlation scatter plot of the expression of miR-181a and IDO mRNA in MSCs derived from normal umbilical cord tissues (n = 9) and from patients with PE umbilical cord tissues (n = 7). Abbreviation: IDO, indoleamine 2,3-dioxygenase; PE, preeclampsia.

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Unfortunately, despite the fact that IDO mRNA level increased 10-fold, the protein was undetectable by Western blot or ELISA. Previous reports showed that human MSCs do not constitutively express IDO, but that IDO protein expression is induced by treatment with interferon (IFN)-γ [34]. Moreover, IFN-γ treatment of MSCs stimulates IDO enzyme activity in a dose-dependent manner. Thus, pre-miR-181a transfected MSCs were treated with increasing amounts of IFN-γ for 24 hours after transfection, and IDO mRNA and protein levels were detected using qRT-PCR (Fig. 3D) and Western blot (Supporting Information Fig. 8). Transfection with pre-miR-181a resulted in significant, dose-dependent elevation of IDO expression compared to control in low IFN-γ concentrations but lost its enhanced ability in response to high IFN-γ concentrations.

Consistent with these findings, basal mRNA expression levels of IL-6 and IDO in MSCs derived from PE patients' umbilical cord tissues were both strongly correlated with basal miR-181a levels. Basal mRNA expression levels of IL-6 in MSCs derived from normal term pregnancy umbilical cord tissues were also correlated with basal miR-181a levels. However, basal mRNA expression levels of IDO in MSCs derived from normal term pregnancy umbilical cord tissues were not correlated with basal miR-181a levels (Fig. 3E, 3F). It shows IL-6 may have a stronger correlation with miR-181a than IDO in vivo.

miR-181a Regulates Immunosuppressive Properties of MSCs Through MAPK Signaling Pathway

As IL-6 and IDO are not direct targets of miR-181a, we sought to identify upstream pathways that might be affected by miR-181a overexpression. We used SB431542, a TGF-β pathway inhibitor, to determine if upregulation of IL-6 and IDO was dependent on TGF-β signaling. Inhibition of TGF-β signaling did not alter on IDO expression and only have a modest inhibition on IL-6 expression (Fig. 4A), which shows that upregulation of IL-6 and IDO was independent on TGF-β signaling.

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Figure 4. miR-181a regulates the immunosuppressive properties of mesenchymal stem cells (MSCs) through the mitogen-activated protein kinases (MAPK) signaling pathway. (A): Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of IL-6 and IDO expression 24 hours after treatment with DMSO or SB431542 (at the indicated concentrations) (n = 3). (B, C): MSCs were transfected with pre-miR-181a, and, 24 hours later, with the Erk1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP600125, the STAT1 inhibitor fludarabine, or the nuclear factor-κB (NF-κB) inhibitor PDTC for another 24 hours. qRT-PCR was used to analyze the expression of IL-6 (B) and IDO (C) after treatment (n = 3). (D): Western blot was used to detect MAPK pathway activation after pre-181a transfection. MSC lysates were collected 18, 24, 36, and 48 hours after transfection with pre-181a. Standard Western blot analysis with antibodies specific for phosphorylated and total p38 proteins, phosphorylated and total ERK1/2 proteins, phosphorylated and total JNK proteins and β-tubulin. Abbreviations: DMSO, dimethylsulfoxide; IDO, indoleamine 2,3-dioxygenase; JNK, c-Jun NH2-terminal kinase; PDTC, pyrrolidinedithiocarbamate.

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Previous studies reported that IL-6 and IDO expression are regulated by MAPK, NF-κB, and STAT1 pathways [35–39]. Based on this knowledge, we used MAPK, NF-κB, and STAT1 pathway inhibitors to determine whether upregulation of IL-6 and IDO via miR-181a was dependent on these pathways. We found that application of a JNK inhibitor blocked pre-miR-181a-induced IDO mRNA expression, and application of a p38 inhibitor blocked pre-miR-181a-induced IL-6 mRNA expression (Fig. 4B, 4C). Erk, NF-κB, and STAT1 inhibitors did not affect expression of IL-6 or IDO. Besides, p38 inhibitor also blocked pre-miR-181a-induced IL-6 protein expression (Supporting Information Fig. 9). These data indicate that miR-181a-induced expression of IL-6 and IDO is dependent on MAPK signaling. Then Western blot was performed to detect the activation of MAPK pathway. The increased activity of JNK and p38 pathways was both detected 18 hours after pre-miR-181a transfection and reach peak values 36 hours after transfection. Erk pathway activity was also detected and reached peaked 18 hours after transfection (Fig. 4D).

miR-181a Attenuates Immunosuppressive Properties of MSCs In Vitro

It is known that MSCs are able to suppress T-cell activation. In order to determine whether overexpression of miR-181a has any affect on the immunosuppressive properties of MSCs, purified T cells were stimulated with PHA in the presence or absence of MSCs transfected with either pre-miR-181a or with pre-nc. After 24 and 72 hours, T cells were harvested, and proliferation was measured using [3H] thymidine incorporation. The results demonstrated that, after 72 hours of coculture, MSCs transfected with pre-miR-181a and MSCs transfected with pre-nc both inhibited mitogen-induced T-cell proliferation. Surprisingly, after 24 hours of coculture, MSCs transfected with pre-miR-181a, but not control transfected MSCs, enhanced the proliferation of T cells in response to PHA treatment at higher MSCs:T ratio (1:5 and 1:10) (Fig. 5A, 5B).

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Figure 5. miR-181a attenuates the immunosuppressive properties of MSCs in vitro. T-cells were cocultured with pre-nc transfected MSCs or pre-181a transfected MSCs. All T-cells were activated with PHA/IL-2, except control. A range of MSCs:T-cell ratios (1:5, 1:10, 1:20, and 1:50) were used to perform the mixed lymphocyte reaction (MLR). T-cell proliferation was assessed by [3H] thymidine incorporation assay after 24 hours (A) or 72 hours (B) of culture. PHA/IL-2-activated T-cells were also cultured for 24 hours (C) or 72 hours (D) with pre-nc transfected MSCs or pre-181a transfected MSCs at MSCs:T-cell ratios (1:10) in the presence or absence of IL-6 neutralizing Ab or IDO inhibitor (1-MT) (n = 4). (E): T-cells activated with or without PHA/IL-2 were cultured with pre-nc transfected MSCs or pre-181a transfected MSCs at MSCs:T-cell ratios (1:10). After 24 hours, CD8+ cells were analyzed for intracellular IFN-γ. For IFN-γ staining, T-cells were treated with Brefeldin A for the last 4 hours of cultures. Cells were permeabilized and the proportion of CD8+/IFN-γ+ T-cells was quantified (n = 4). Abbreviations: CPM, counts per minute; IDO, indoleamine 2,3-dioxygenase; MSCs, mesenchymal stem cells; PHA, phytohaemagglutinin.

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We showed that miR-181a enhanced IL-6 and IDO expression in MSCs. To investigate the importance of these soluble factors, anti-IL-6 neutralizing Ab and 1-M-Trp, an inhibitor of IDO enzymatic activity, were added to the cultures. We found that, after 24 hours of coculture, MSCs with anti-IL-6 neutralizing Ab enhanced proliferation of T cell in response to mitogen treatment at MSCs:T ratio of 1:10. The presence of anti-IL-6 did no effect the long-term coculture. MSCs transfected with miR-181a and in the presence of 1-M-Trp enhanced proliferation of T cell after 24 and 72 hours of coculture (Fig. 5C, 5D), as compared to control.

We next investigated whether MSCs affect expression of markers of T-cell activation. T cells were harvested after 24 hours of coculture, and CD8+ cells were analyzed for the expression of CD25, CD69, and intracellular IFN-γ. After T cells were in contact with MSCs, IFN-γ production was impaired (Fig. 5E), expression of CD25 and CD69 were only slightly affected (Supporting Information Fig. 10). However, compared with T cells that were in contact with MSCs transfected with pre-nc, IFN-γ production was enhanced in T cells that were in contact with MSCs transfected with pre-miR-181a. Expression of miR-181a did not alter expression of CD25 and CD69. The ability of MSCs to suppress T-cell proliferation was attenuated by miR-181a and required IL-6. miR-181a also attenuated the ability of MSCs to suppress T-cell IFN-γ production. Thus, the ability of MSCs to suppress T-cell activation was attenuated by miR-181a and partly required IL-6.

miR-181a Expression Impairs MSC-Based Therapy for Experimental Colitis Induced by Dextran Sulfate Sodium

We next determined the in vivo effects of miR-181a on MSC-based therapy. The immunosuppressive and anti-inflammatory effects of MSCs have been demonstrated in the treatment of several animal disease models, including graft-versus-host disease, diabetes, rheumatoid arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, periodontitis, inflammatory bowel disease (IBD), and sepsis [32]. Our study proved that systemic infusion with MSCs has beneficial effects on healing in a mouse model of PE induced by adoptive transferring of activated Th1 cells (Supporting Information Fig. 11). Although Th1 cell-induced PE models have features of PE, this model is still a poor overall model of the human disease and is not a widely used model because it does not show the complete spectrum of pathophysiological changes associated with PE. Therefore, the PE animal model was not used to investigate the immunosuppressive and repair capacity of MSCs in vivo.

Recently, several groups have reported that treatment with human MSCs can attenuate the progression of experimental colitis induced by DSS [32, 40, 41]. We confirmed that oral administration of 3% DSS for 8 days (Fig. 6A) induced acute colitis in C57BL/6 mice characterized by an overall elevation of colitis scores based on the presence of sustained weight loss (Fig. 6B), bloody diarrhea/loose feces (Fig. 6C). DSS-treated mice also had shorter colons (Fig. 6D), lighter colons (Fig. 6E), and a higher mortality rate (Fig. 6F) than control mice. Histological studies revealed severe colonic transmural inflammation, increased wall thickness, localized inflammatory cell infiltration, epithelial ulceration with degeneration of crypt architecture, and loss of goblet cells (Fig. 6G, 6H).

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Figure 6. miR-181a expression attenuates MSC-based therapy of experimental colitis induced by DSS (n = 11). (A): Scheme for the experimental course of experimental colitis induced by DSS and MSC-based therapy. Acute colitis was induced by administering 3% (w/v) DSS (molecular mass 36,000–50,000 Da; MP Biochemicals) in drinking water ad libitum for 8 days. A total of 2 × 106 of pre-nc transfected MSCs or pre-181a transfected MSCs resuspended in 100 μl of phosphate buffer saline (PBS) were i.p. injected into mice 1 and 3 day after initiation of DSS treatment individually. At day 13 after colitis induction, mice were sacrificed by CO2 euthanasia. Clinical progression of the disease was monitored by body weight changes (B), colitis score evaluation (C), and survival rate (F). Body weight change was calculated by dividing the body weight on indicated days by the body weight on day 0 (starting body weight). After mice were sacrificed at day 13, colon length (D) and weight (E) were measured. (G, H): Histopathological analysis of colitis after sacrifice. Abbreviations: MSC, mesenchymal stem cell; DSS, dextran sulfate sodium.

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Systemic infusion with MSCs protected mice against colitis-related tissue injuries and reduced the overall disease severity, shown here as a decrease in disease score, reversal and stabilization of body weight, and suppressing of colonic inflammation. MSC-treated mice had longer, heavier colons, reduced mortality compared with DSS-treated mice on day 12. Histologically, MSCs' treatment significantly reduced colonic transmural inflammation and decreased wall thickness, restored goblet cells and suppressed mucosal ulceration and focal loss of crypts, thus restoring normal intestinal architecture.

Importantly, our results showed that miR-181a attenuates the MSC-based protection against colitis-related tissue injuries that is provided to DSS-treated mice. The overall disease scores were not significantly different from DSS-treated mice. Systemic infusion with miR-181a transfected MSCs neither reversed nor stabilized body weight and failed to suppress colonic inflammation. Unlike the control MSC-treated mice, where the gross appearance of colons remained normal, miR-181a transfected MSC-treated mice showed signs of severe acute inflammation. The colons in the miR-181a transfected MSC-treated mice were shorter and lighter than the colons from control MSC-treated mice, although there is no significant difference. Histologically, samples from mice with systemic infusion of miR-181a transfected MSCs were similar to those of DSS-treated mice.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In this study, we analyzed the expression of miR-181a in MSCs derived from umbilical cord and decidua. The results showed that the basal expression of miR-181a was higher in MSCs from PE patients than MSCs from women with normal term pregnancy. Our results confirmed that miR-181a is a regulator of both MSCs' proliferation and immunosuppressive properties. miR-181a prevented the proliferation of MSCs through TGF-β signaling pathway by targeting TGFBR1 and TGFBRAP1, and miR-181a regulated MSCs' immunosuppressive properties through the MAPK signaling pathway. Specifically, miR-181a enhanced IL-6, VEGF, and IDO expression, resulting in attenuation of the MSCs immunosuppressive properties in vitro and in vivo.

miR-181a can regulate TGF-β signaling of MSCs. TGF-β signaling affects multiple cellular processes including proliferation, migration, differentiation, and apoptosis [42–44]. In general, TGF-β is antiproliferative in cells of epithelial origin but promotes growth of mesenchymal cells [45, 46]. Jian et al. [47] reported that TGF-β1 induces proliferation of bone marrow (BM)-MSCs and showed an important role of TGF-β signaling in MSCs proliferation. TβRI is encoded by TGFBR1 gene. The TGF-β ligand binds type II TGF-β receptors (TβRII), which activate the TβRI by promoting phosphorylation within the glycine-serine rich GS region of TβRI [44]. TβRI knockout mice are embryonically lethal due to severe defects in yolk sac and placental vascular development [48]. TRAP1, which encoded by the TGFBRAP1 gene, is a 96-kDa cytoplasmic protein that binds to TGF-β receptors and thought to play a role in TGF-β signaling. TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator Smad4 in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-β signaling in functional assays, deletion of TRAP1 inhibits TGF-β signaling and impairs the interaction between Smad4 and Smad2 [49]. However, TRAP1 does not affect the phosphorylation of Smad2. In order to measure the ability of miR-181a to affect TGF-β signaling through TRAP1 and TGFBR1, we analyzed expression of PAI-1 mRNA, a classic TGF-β1-induced message. The result also showed miR-181a can affect TGF-β signaling. Besides, we also found out that the mRNA expression of TGFBR2 was enhanced by miR-181a. The mechanism of the TGF-β-TβRII-TβRI-Smad2/3 pathway has been well-established. TβRI knockout or TβRI inhibitor-treated cells will block TGF-β-induced phosphorylation of Smad2, which means without TβRI only TβRII cannot induce the activation of TGF-β signaling [50, 51]. Moreover, it was proofed that TGF receptor type I:type II ratio has an important role in the activation of TGF-β signaling and expression of TGF-β-induced genes [52]. Importantly, our results showed that the expression of TGFBR1 and TGFBRAP1 were downregulated and TGFBR2 is upregulated by miR-181a, which cause a change in the TGF receptor type I:type II ratio. Moreover, we found that miR-181a can affect the phosphorylation of Smad2 (activation of TGF-β signaling) (Fig. 1G) and downstream gene expression (PAI-1). These data strongly suggest that miR-181a may regulate TGF-β signaling through TGFBR1 and TGFBRAP1.

The function of miR-181a in regulation of the immune system was first described in T and B cells. miR-181 is highly expressed in thymocytes and its immunosuppressive ability was first detailed in the maintenance of cell differentiation in thymocyte development. Overexpression of miR-181 increases the rate of B-lymphocyte production and decreases the rate of T-lymphocyte production in mice [53]. miR-181a also functions as an intrinsic modulator of T-cell sensitivity and selection by increasing the basal phosphorylation levels of Lck and ERK [21]. However, the function of miR-181a in the regulation of immunosuppressive properties of MSCs has not been well-characterized.

miR-181a regulates immunosuppressive properties of MSCs in independence of IDO. The immunosuppressive properties of MSCs are usually performed by soluble factors and cell-to-cell contract. IDO is thought to be critical for the inhibition of immune cells' proliferation [54, 55], and its production contributes to the immunosuppressive effect of MSCs [56, 57]. Although the basal expression of IDO in MSCs is very low, IDO can be induced by IFN-γ and other inflammatory cytokines [58] and cause thousand times of expression and become functional. Meisel [55] reported that human BM stromal cells inhibit allogenic T-cell proliferation through IDO-mediated tryptophan degradation. Interestingly, our results showed that miR-181a enhanced both IDO mRNA expression through activating JNK pathway in MSCs and IFN-γ induced IDO expression in low IFN-γ concentration, however, miR-181a could not potentiate the immunosuppressive properties of MSCs by IDO. One possible explanation is that endogenous IFN-γ levels are not low enough to facilitate effective regulation by miR-181a. Our results support the possibility. We found that when the concentration of stimulating IFN-γ is higher than 500 U/ml, miR-181a transfected MSCs and pre-nc transfected MSCs have equivalent IDO expression. Another possible explanation is that the tryptophan in short-term coculture (24 hours) media has not been totally degraded by IDO, since IDO inhibit immune cells through IDO-mediated tryptophan degradation. At this time point, IL-6 may play a key role in the regulation of T-cell proliferation. But after a long-term coculture (72 hours), tryptophan has already been entirely degraded, and at this time point, IDO may play a key role in the regulation of T-cell proliferation. Additionally, as the expression of IDO is dependent on IFN-γ, and because T-cells can express IFN-γ, IDO might function in the regulation of T-cells' proliferation. In the future, it would be interesting to see whether MSCs has any antiproliferative effect when cocultured with B cells, which do not express IFN-γ.

IL-6 is necessary for miR-181a enhancing capability of MSCs to T-cell proliferation. IL-6 is also an important soluble factor with a wide range of biological activities in immune regulation, hematopoiesis, inflammation, and oncogenesis. Over the years IL-6 has been assigned both proinflammatory and anti-inflammatory characteristics. However, the literature does not universally define IL-6 as being selective for the induction of a Th1- or Th2-type response. IL-6 should be defined as a resolution factor that balances proinflammatory and anti-inflammatory outcomes to further the immunological response [59]. There is also a controversy whether IL-6 have a role in the immunosuppressive potential of MSCs. Eddahri et al. [60] reported that IL-6 promotes the differentiation of naïve T lymphocytes into helper cells. MSCs constitutively produce IL-6. Some studies reported that IL-6 may play a major role in the MSC-mediated inhibition of dendritic cell (DC) differentiation [61, 62], while other studies showed that IL-6 have no effect in the MSC-mediated inhibition of DC differentiation [63, 64]. It might be true that IL-6 may play a role in the MSC-mediated inhibition of DC differentiation. But whether IL-6 have affect in the MSC-mediated inhibition of lymphocyte proliferation and activation have not been fully investigated. Najar Mehdi reported that MSCs promote the proliferation of T lymphocytes and IL-6 is, in part, responsible for supporting T-cell proliferation [65]. Therefore, it seems that IL-6 might be a proinflammatory factor in MSCs. In this study, surprisingly, we found that miR-181a enhanced IL-6 expression in MSCs. To determine whether overexpression of miR-181a, and thus IL-6, in MSCs has any affect on the immunosuppressive properties of MSCs, purified T-cells were cocultured with MSCs. Interestingly, the results showed the ability of MSCs to suppress T-cell activation was enhanced by short-term miR-181a expression but was not changed by long-term miR-181a expression in vitro. Moreover, an anti-IL-6 neutralizing Ab blocked the ability of MSCs to enhance T-cell proliferation, while addition of 1-M-Trp to the culture enhanced the ability of MSCs to promote T-cell proliferation. These data indicated that IL-6 is necessary for miR-181a enhancing capability of MSCs to T-cell proliferation.

MiRs are always the negative regulators of their target gene's protein expression. IL-6 are not direct targets of miR-181a and previous studies reported that IL-6 expression are regulated by MAPK, NF-κB, and STAT1 pathways [35–39]. Based on this knowledge, we used MAPK, NF-κB, and STAT1 pathway inhibitors to determine whether upregulation of IL-6 via miR-181a was dependent on these pathways. We found that application of a p38 inhibitor, other than JNK inhibitor, blocked pre-miR-181a-induced IL-6 mRNA and protein expression. Erk, NF-κB, and STAT1 inhibitors did not affect expression of IL-6. These data indicate that miR-181a-induced expression of IL-6 is dependent on p38 signaling.

The phosphorylation of MAPK signaling is mostly negatively regulated by “protein-tyrosine phosphatase”(PTP) and “dual specificity phosphatase” (DUSP). miR-181a was reported to increase the basal phosphorylation level of ERK by targeting genes encoding DUSP5 and DUSP6. These phosphotases dephosphorylate both threonine and tyrosine residues. We analyzed the predicted targets of miR-181a to identify DUSP family members and found that, not only DUSP5 and DUSP6, but also DUSP3, DUSP8, and DUSP10 may be targeted by miR-181a. Interestingly, DUSP8 and DUSP10 bind JNK and p38 substrates related to JNK and p38 pathway activation [66]. Thus, we speculate that miR-181a may regulate the activation of JNK and p38 by targeting DUSP8 and DUSP10. Besides, we also found out some PTPs, which are the predict targets of miRNA-181a, might affect the phosphorylation of JNK and p38 including PTPN9 [67], PTPRE [68], PTPN2 [69], PTPN22 [21], and PTPN3 [70], and those PTPs may also affected by miR-181a to regulate the activation of JNK and p38.

The immunosuppressive and anti-inflammatory effects of MSCs have been demonstrated in the treatment of several animal models of disease, including graft-versus-host disease, diabetes, rheumatoid arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, periodontitis, IBD, and sepsis [7]. Importantly, our study proved that systemic infusion with MSCs has beneficial effects in a mouse model of PE induced by adoptive transferring of activated Th1 cells (Supporting Information Fig. 11). Adoptively transferring activated BALB/c Th1-like splenocytes into allogeneically pregnant BALB/c female mice during late gestation cause PE-like symptoms in pregnant mice characterized by increased blood pressure, fetal rejection, and proteinuria (Supporting Information Fig. 11B–11D) accompanied by glomerulonephritis and massive placental hemorrhage (Supporting Information Fig. 11G, 11H). Histological studies revealed while normal pregnant animals show no pathological signs in the kidneys as representatively, fibrosis and glomerular disorganization could also be observed in animals with Th1 cells adoptively transferred. Th1 cells adoptively transferred mouse also show large areas of massive hemorrhage in placenta (Supporting Information Fig. 11E, 11H). Our results showed that systemic infusion with MSCs protected mice against Th1-induced PE, which revealed here a decrease in blood pressure, proteinuria, and fetal rejection rate (compared with Th1 cells adoptively transferred mouse at day 14). Histologically, MSCs' infusion also reversed the condition of PE in kidney and placenta. These findings suggest that cell-based therapy using MSCs can ameliorate Th1-induced PE in mice, while miR-181a attenuates the MSC-based protected mice against Th1-induced PE.

Although the Th1 cell-induced PE model has features of PE, it is not a widely used mouse model. MSCs hold an immunoregulatory capacity and elicit immunosuppressive effects in a number of situations. Several studies have reported the treatment with human BM- or adipose-derived MSCs in DSS-induced experimental colitis. Therefore, an experimental colitis model was used to examine the role of miR-181a in MSC-based therapy. In this study, UCMSCs were used to treat DSS-induced experimental colitis. The results indicated that UCMSCs effectively treat DSS-induced experimental colitis. Expectedly, miR-181a expression attenuates this MSC-based therapy. It is likely that miR-181a expression impairs treatment by limiting the quantity of MSCs and rendering them less able to prevent immune cell activation in vivo.

In addition, maternal plasma IL-6 levels are elevated in women with PE [71, 72], and increasing IL-6 levels can stimulate production of agonistic auto-antibodies to the angiotensin II type 1 receptor. The source of increasing IL-6 in maternal plasma remains unknown [73, 74], but several studies found that the placenta is not the source. Our research showed that miR-181a induced expression of IL-6 in MSCs but not in trophoblast cells (data not show). Therefore, highly expressed miR-181a in MSCs may be a source of elevated IL-6 levels in maternal plasma in women with PE.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In conclusion, MSCs have an important role in immune balance at the maternal–fetal interface. Elevated miR-181a expression at the maternal–fetal interface prevents the proliferation of MSCs and attenuates their immunosuppressive properties. Thus, PE may be the ultimately result of miR-181a misregulation. These findings will aid in our understanding of miRNA function in MSCs and may provide an opportunity for potential novel therapies to target PE.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work supported by grants from the National Natural Science Foundation (project number: 81072410), the Special Research Grant of Jiangsu Province Department of Health (project number: XK200709 and JHB2011-1), and the Special grant for maternal-fetal medicine from Jiangsu province Health Department of China (project number: 81070508).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
SC_12-0204_sm_supplFigure1.tif606KSupplementary Figure 1. miR-181a does not affect phenotypic surface antigens of MSCs. Flow cytometry characterization of human MSCs 48 h after transfection with control (pre-nc and anti-nc), pre-miR-181a (A) and anti-miR-181a (B) individually. Expression of MSCs phenotypic surface antigens (CD105, CD73, CD90, HLA-ABC, CD29, CD44, CD45, CD106, HLA-DR, CD19, CD11b, CD14, CD34, CD31) was detected using FACS (n =3).
SC_12-0204_sm_supplFigure2.tif117KSupplementary Figure 2. PE patient derived MSCs amplified more slowly than normal patient derived MSCs. Cell growth curve of MSCs derived from PE (n=20) and normal (n=20) patients. MSCs of passage 2 were plated in 24-well plates in DF12 supplemented with 10% FBS at a density of 5×103 cells/well. The cells were detached with 0.25% trypsin-EDTA and counted every 2 days and until day 14. Bars show the mean. P values were determined by Mann-Whitney test with Bonferroni correction. P<0.05.
SC_12-0204_sm_supplFigure3.tif239KSupplementary Figure 3. miR-181a does not affect MSCs apoptosis. MSCs were transfected with control (pre-nc or anti-nc), pre-miR-181a and anti-miR-181a individually. 30 pmol of pre-miR-181a or anti-miR-181a were used for transfection in 12-wells plates at a density of 8×104 cells/well. 48 h after transfection, MSCs were collected for the following experiment. Effects of miR-181a on MSCs discriminated by Annexin- V/PI doublestain 48 h after transfection (n =3). Data are representative of three independent experiments.
SC_12-0204_sm_supplFigure4.tif53KSupplementary Figure 4. (A) Phosphorylation of Smad2 after SB431542 treatment. Whole-cell lysates of untreated MSCs or MSCs treated with TGF-β1 and the type I TGF-β receptor inhibitor SB431542 for 30 min. Phosphorylation of Smad2 was detected by western blot with a polyclonal antiphospho-Smad2 antibody. (B) MSCs proliferation after 48 h of SB431542 treatment was determined by Cell Counting Kit (n =4).
SC_12-0204_sm_supplFigure5.tif33KSupplementary Figure 5. qRT-PCR analysis of PAI-1 expression in pre-181a transfected MSCs after 1 h of treatment with TGF-β1 (n =3). Data are representative of three independent experiments.
SC_12-0204_sm_supplFigure6.tif55KSupplementary Figure 6. qRT-PCR (A) and western blot (B) analysis of the expression of the TGFBR1 and TGFBRAP1 48 h after anti-181a transfection (n =3).
SC_12-0204_sm_supplFigure7.tif45KSupplementary Figure 7. (A) qRT-PCR analysis of the expression of IL-6 and IDO 48 h after anti-181a transfection (n =3). (B) IL-6 protein expression, as detected by ELISA 48 h after anti-181a transfection (n =3).
SC_12-0204_sm_supplFigure8.tif50KSupplementary Figure 8. Pre-181a transfected MSCs and pre-nc transfected MSCs were treated with indicated concentrations (0 U, 200 U, 500 U, 1000 U per milliliter) of IFN-γ for 72 h and then cells were harvested and IDO protein was detected by western blot.
SC_12-0204_sm_supplFigure9.tif41KSupplementary Figure 9. p38 inhibitor blocked pre-miR-181a induced IL-6 protein expression. MSCs were transfected with pre-miR-181a, and, 24 h later, with the Erk1/2 inhibitor PD98059, the p38 inhibitor SB203580, the JNK inhibitor SP600125, the STAT1 inhibitor fludarabine, or the NF-κB inhibitor PDTC for another 24 h. The protein level of IL-6 expressions were detected by ELISA 48h after pre-181a transfection. (n =3)
SC_12-0204_sm_supplFigure10.tif421KSupplementary Figure 10. T cells activated with or without PHA/IL-2 were cultured with pre-nc transfected MSCs or pre-181a transfected MSCs at MSCs: T cell ratio of 1:10. After 24 hours, CD8+ cells were analyzed for expression of activation molecules using anti-CD25 or anti-CD69 monoclonal antibodies. Cells were permeabilized and the proportion of CD8+/CD69+ and CD8+/CD25+ T cells were quantified (n =4).
SC_12-0204_sm_supplFigure11.tif1149KSupplementary Figure 11. miR181a attenuates benefits MSCs have on healing in a mouse model of preeclampsia induced by adoptive transfering of activated Th1 cells. (A) Scheme for the experimental course of preeclampsia model induced by activated Th1 cells and MSCs-based therapy. Blood pressure (B) and proteinuria (D) were detected on day on DG14. (C) The fetal rejection rate in pregnant mice after PBS treatment (n=11) or transfer of activated Th1-like cells (n=8) or transfer of activated Th1-like cells and MSCs (n=5) or transfer of activated Th1-like cells and miR-181a transfected MSCs (n=5), as calculated from the ratio of rejected fetuses to total implantation sites. (E,F) Representative picture of placenta and the fetuses from a pregnant mouse. After transfer of activated Th1-like cells or transfer of activated Th1- like cells and miR-181a transfected MSCs, some placenta and fetuses showed placental bleeding (red arrow). (G) Histopathological analysis of kidney after sacrificed. Representative examples of abnormal cell distribution as well as cell clusters in the glomerulum of pregnant cell recipients; normal pregnant animals show no pathological signs in the kidneys. Moreover, fibrosis and glomerular disorganization could also be observed in animals with PE-like signs. MSCs infusion reversed the condition of PE. However, infusion with pre-181a transfected MSCs can not reverse the condition of PE. (H) Histopathological analysis of placenta after sacrificed, animals that received Th1 activated cells and developed PE show large areas of massive hemorrhage (blood), animals that received Th1 activated cells and infusion with pre-181a transfected MSCs also show some areas of massive hemorrhage (blood).

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