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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

MicroRNA is a family of noncoding RNAs that exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. MicroRNA-15a (miR-15a) is reported to induce cell apoptosis by negatively regulating the expression of Bcl-2, which suppresses the apoptotic processes. The purpose of this study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by cells and could induce Bcl-2 dysfunction and cell apoptosis in the synovium of arthritic mice in vivo.

Methods

Autoantibody-mediated arthritis was induced in male DBA/1J mice. In the experimental group, double-stranded miR-15a labeled with FAM–atelocollagen complex was injected into the knee joint. In the control group, control small interfering RNA–atelocollagen complex was injected into the knee joint. Synovial expression of miR-15a was analyzed by quantitative polymerase chain reaction, FAM by fluorescence microscopy, Bcl-2 by Western blotting, and Bcl-2 and caspase 3 by immunohistochemistry.

Results

The expression of miR-15a in the synovium of the experimental group was significantly higher than that in the control group. Green fluorescence emission of FAM was observed in the synovium of the experimental group. Bcl-2 protein was down-regulated and the expression of caspase 3 was increased as compared with that in the control group.

Conclusion

These results indicate that the induction of cell apoptosis after intraarticular injection of double-stranded miR-15a occurs through inhibition of the translation of Bcl-2 protein in arthritic synovium.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and excessive inflammatory cell infiltration in the joints, leading to erosion of the articular cartilage and bone margins, with subsequent destruction of the joint (1). Despite an explosion of studies on inflammation over the last 2 decades, the detailed mechanism of synovial hyperplasia and inflammation is not well understood. Recently, a rapid proliferation of synovial cells, overexpression of inflammatory genes, and impairment of apoptosis were noted to play a role in the persistence of abnormal cells involved in the disease process (2–4). Several studies have shown that an antiapoptotic protein, particularly in Bcl-2, is highly up-regulated in RA synovial fibroblasts as compared with osteoarthritis synovial fibroblasts and that the apoptotic process in RA synovial fibroblasts may be suppressed by the overexpression of Bcl-2 (5–7).

The microRNA (miRNA) are a family of noncoding RNAs that are believed to be important in many biologic processes through regulation of gene expression. Many miRNA are evolutionarily conserved across phyla. MicroRNA are single-stranded RNA molecules of ∼22 nucleotides in length. They play a crucial role in the regulation of gene expression by inhibiting protein translation or degradation of target messenger RNA (8). MicroRNA exhibit tissue-specific or developmental stage–specific patterns of expression and are associated with human diseases, such as cancer, leukemia, and viral infections (9, 10). Depending on the disease, the expression of miRNA is up-regulated in some conditions and down-regulated in others (11, 12). It is therefore possible that miRNA might be a novel therapeutic target for human diseases.

The results of several therapeutic trials examining the regulation of endogenous miRNA that are related to disease pathogenesis through in vivo administration of specific antisense oligoribonucleotides or double-stranded miRNA have been reported (13–15). Recently, miRNA-15a (miR-15a) was reported to down-regulate the expression of Bcl-2 by inhibiting protein translation, which leads to cell apoptosis (16). It was therefore hypothesized that the overexpression of miR-15a in the synovium plays a role in the treatment of RA by inducing cell apoptosis in the synovium due to Bcl-2 dysfunction.

The purpose of the present study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by the cells in vivo, thus inducing cell apoptosis, in the synovium of arthritic mice.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All procedures were performed according to the Guidelines for Animal Experimentation, Hiroshima University, and with the approval the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University.

Preparation of double-stranded miR-15a and small interfering RNA (siRNA)–atelocollagen complex.

We used double-stranded miR-15a that was designed for intraarticular injection in the experimental group (sequences 5′-UAG-CAG-CAC-AUA-AUG-GUU-UGU-G-3′ and 5′-CAG-GCC-AUA-UUG-UGC-UGC-CUC-A-3′ labeled with FAM; Hokkaido System Science, Sapporo, Japan). Control siRNA with no specific function were also prepared for use as a control group (sequences 5′-ATC-CGC-GCG-ATA-GTA-CGT-A-3′ and 3′-overhung dTdT/dTdT [sense/antisense] siRNA negative control; B-Bridge International, Mountain View, CA).

Atelocollagen is a highly purified type I collagen isolated from calf dermis by pepsin treatment (Koken, Tokyo, Japan). The double-stranded miR-15a and atelocollagen complex was prepared as follows. An equal volume of atelocollagen (in phosphate buffered saline, pH 7.4) and miRNA solution (10 μg/10 μl) was combined and mixed by rotation at 4°C for 20 minutes. The nonspecific control siRNA and atelocollagen complex were prepared in the same way.

Animals.

Male DBA/1J mice (Japan SLC, Tokyo, Japan) ages 7–9 weeks were used in these experiments. Mice were housed at the Laboratory Animal Center of Hiroshima University under standard diurnal conditions of light/dark and were fed a standard commercial diet and given tap water ad libitum. Arthritis was induced by an arthritogenic cocktail of 4 monoclonal antibodies (mAb) to type II collagen (Chondrex, Redmond, WA) combined with lipopolysaccharide simulation according to the method of Terato et al, as previously described (17, 18). The mice were injected intravenously with 2 mg of mAb on day 0, followed by intraperitoneal injection of 50 μg of lipopolysaccharide on day 2. Mice were then monitored daily for the development of arthritis, beginning on the day after the first mAb injection. The onset of clinically distinct arthritis was observed on day 4, and the experimental group was given a single intraarticular injection of double-stranded miR-15a–atelocollagen complex on day 9. Control mice were injected with control siRNA.

Injection of miRNA or siRNA–atelocollagen complex into the mouse knee joint.

On day 9, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (1 ml/kg). The skin was longitudinally incised on the center of the right knee joint, and the capsule and patellar tendon were exposed. The mice in the experimental group received an intraarticular injection of double-stranded miR-15a–atelocollagen complex (10 μg of miR-15a/20 μl) into the right knee joint. The mice in the control group received an injection of control siRNA–atelocollagen complex (10 μg of nonspecific control siRNA/ 20 μl). After injection, the incised skin was sutured, and the mice were returned to their cages and were free to exercise thereafter.

After 24 hours, some mice were killed, and the knee joints and internal organs were harvested. After 3 days, the remaining mice were killed, and the knee joints were harvested. For the polymerase chain reaction (PCR) analysis, total RNA was isolated from synovium harvested from the knee joint and internal organs (liver, lung, spleen, kidney, and heart) using TRIzol reagent (Invitrogen, Carlsbad, CA). For histologic analysis, the knee joint was quickly embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC), snap-frozen in liquid nitrogen, and stored at −80°C. For Western blotting, synovium from the knee joint was excised, and protein was isolated using T-PER tissue protein extraction reagent (Pierce, Rockford, IL).

Quantitative reverse transcription–PCR (RT-PCR).

Ten nanograms of total RNA was used to synthesize single-stranded complementary DNA using a TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA). Initially, 7 μl of RT master mix, 3 μl of primer, and 5 μl of RNA (10 ng) were mixed. For the RT procedure, the reaction mixture was incubated at 16°C for 30 minutes, at 42°C for 30 minutes, and then heated at 85°C for 5 minutes.

For target amplification, 1.33 μl of product from the RT reaction, 1 μl of TaqMan miRNA assay primer and probe mixture, 10 μl of TaqMan 2× Universal PCR Master Mix, and nuclease-free water were mixed. Reactions were performed with a thermal cycler (Bio-Rad, Richmond, CA). The reactions were heated at 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C.

To quantify the expression levels of miR-15a and small nucleolar RNA-135 (snoRNA-135), we used the TaqMan miRNA assays mmu-miRNA-15a and snoRNA-135 (Applied Biosystems), respectively. The expression of miR-15a was defined from the threshold cycle (Ct), and the relative expression levels were calculated according to a previously described method (19), after normalization with reference to the expression of snoRNA-135. The average value in the control group was set at 1 and was used to calculate the fold change in the target gene.

Histologic and immunofluorescence analyses.

The knee joints embedded in compound were cut into 6-μm serial sections in the sagittal plane. For fluorescence microscopy of FAM-labeled miRNA-15a, 6-μm serial sections were mounted on saline-coated glass slides, air-dried, and fixed with 4.0% paraformaldehyde at 4°C for 5 minutes. Then, 4′,6-diamidino-2-phenylindole (DAPI) solution was applied for 5 minutes for nuclear staining.

For immunofluorescence staining of Bcl-2 and caspase 3, 6-μm serial sections were mounted on saline-coated glass slides, air-dried, fixed with 4.0% paraformaldehyde at 4°C for 5 minutes, and immediately stained. For histologic detection of Bcl-2 expression and cell apoptosis, immunohistochemistry was performed with mouse anti–Bcl-2 mAb (Upstate Biotechnology, Lake Placid, NY) and with rabbit polyclonal anti–active caspase 3 antibody (Promega, Madison, WI) to detect apoptotic cells. The secondary antibodies for each immunostaining were Alexa Fluor 488–conjugated or Alexa Fluor 568–conjugated goat anti-mouse IgG for Bcl-2 and Alexa Fluor 488–conjugated or Alexa Fluor 568–conjugated goat anti-rabbit IgG for caspase 3 (all from Molecular Probes/Invitrogen, Carlsbad, CA). DAPI solution was applied for 5 minutes to detect nuclear staining.

Western blotting for Bcl-2.

Two micrograms of protein was separated on NuPAGE Novex Bis-Tris Mini Gels and transferred onto a nitrocellulose membrane (both from Invitrogen). Mouse anti–Bcl-2 mAb and rabbit polyclonal antiactin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Horseradish peroxidase–conjugated goat anti-mouse IgG for Bcl-2 and goat anti-rabbit IgG for actin (both from MP Biomedicals, Santa Ana, CA) were used as secondary antibodies. Band detection was performed using enhanced chemiluminescence reagent (ECL Western Blotting Detection Reagents; GE Healthcare UK, Little Chalfont, UK).

Statistical analysis.

The Mann-Whitney U test was used to compare gene expression between 2 groups. P values less than 0.05 were considered statistically significant. All statistical analyses were performed on a personal computer using the StatView version 5.0 statistical package (Abacus Concepts, Berkeley, CA).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Down-regulation of miR-15a and up-regulation of Bcl-2 in the synovium of arthritic mice.

To confirm the expression of endogenous miR-15a in the synovium, we performed quantitative RT-PCR. The expression of mmu-miR-15a was significantly down-regulated in arthritic mice as compared with nonarthritic mice (Figure 1A). In addition, the up-regulation of Bcl-2 protein in arthritic mice as compared with nonarthritic mice was confirmed by Western blotting (Figure 1B).

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Figure 1. Down-regulation of microRNA-15a (miR-15a) and up-regulation of Bcl-2 in the synovium of mice with antibody-mediated arthritis. A, Expression of miR-15a in the synovium of arthritic and nonarthritic mice on day 9 after injection of monoclonal antibody, as determined by quantitative reverse transcription–polymerase chain reaction. The average value for miR-15a in nonarthritic mice was set at 1, and the relative amounts of miR-15a in arthritic mice were plotted as the fold induction. Values are the mean and SD of 5 mice per group. = P < 0.05. B, Immunoblots showing the accumulation of Bcl-2 in arthritic, but not nonarthritic, mice. Actin was used as the loading control.

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Function of double-stranded miR-15a in the synovium of arthritic mice.

To confirm that the double-stranded miR-15a administered by intraarticular injection could be taken up by the cells in the synovium, quantitative RT-PCR was used to detect the injected double-stranded miR-15a, and frozen sections were prepared to examine the fluorescence of the FAM-labeled double-stranded miR-15a. The level of miRNA-15a was significantly higher in the experimental group (mean ± SD 3.34 ± 1.76–fold) in comparison to the control group (1.00 ± 0.88–fold) (Figure 2A).

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Figure 2. Expression and function of double-stranded microRNA-15a (miR-15a) in the synovium of arthritic mice following intraarticular injection. A, Gene expression of miR-15a in the synovium following intraarticular injection into the knee joint. Expression of miR-15a was significantly increased, as determined by reverse transcription–polymerase chain reaction. Values are the mean and SD of 6 mice per group. = P < 0.05. B, Immunofluorescence analysis of the synovium 24 hours after injection of miR-15a or control small interfering RNA (siRNA) into the knee joint. No green fluorescence is seen in the synovium of joints injected with control siRNA (a and b), whereas green fluorescence is evident in the synovium of joints injected with FAM-labeled double-stranded miR-15a (c and d), as indicated on images obtained under brightfield (a and c) and darkfield fluorescence (b and d) microscopy. Arrows in d indicate strong green fluorescence in the synovium. High-power field views of miR-15a–injected joints show staining with FAM (e), 4′,6-diamidino-2-phenylindole (DAPI) (f), and FAM plus DAPI merged (g). The image shown in h is a higher-magnification view of the boxed area in g. C, Western blots showing Bcl-2 expression following intraarticular injection of miR-15a. The expression of Bcl-2 was reduced in joints injected with miR-15a as compared with controls. Actin was used as the loading control. D, Immunohistochemical analysis of Bcl-2 expression in the synovium 24 hours after injection of miR-15a or control siRNA into the knee joint. Bcl-2 expression was reduced after miR-15a injection (b) as compared with control siRNA injection (a), as indicated by Bcl-2 staining (left) and the merged image of Bcl-2 plus DAPI (right). Asterisks in B indicate the synovium; asterisks in D indicate the joint cavity. Bars = 200 μm in B, parts a–d; 100 μm in B, parts e–g; 100 μm in D, parts a and b.

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In frozen sections, no fluorescence was observed in the synovium from the knee joints of control mice. In synovium of the knee joints of mice in the experimental group, however, green fluorescence emission was observed. Specifically, a strong expression of green fluorescence was observed near the joint cavity in knees from the experimental group (Figure 2B, parts b and d). In high-power views, the strong green fluorescence was observed in the cytoplasm of cells in the synovium from mice in the experimental group (Figure 2B, parts e–h). Figure 2B, parts a and c, show the corresponding brightfield images for comparison.

To confirm whether the injected double-stranded miR-15a effectively functioned in the synovium, we examined the levels of Bcl-2 protein, which has been reported to be the direct target of miR-15a. Western blotting showed the Bcl-2 protein level to be down-regulated in the experimental group as compared with the control group (Figure 2C). Immunohistochemistry demonstrated that Bcl-2 expression in the synovium was also suppressed 24 hours after intraarticular injection of the miRNA-15a–atelocollagen complex, especially near the joint cavity (Figure 2D).

Induction of cell apoptosis in the synovium by intraarticular injection of double-stranded miR-15a.

To examine cell apoptosis induced by the injected double-stranded miR-15a, we evaluated the immunohistochemistry of caspase 3. Three days after intraarticular injection, the expression of caspase 3 increased near the joint cavity in the synovium of mice in the experimental group. In contrast, the expression of caspase 3 could not be observed in the control group mice (Figure 3).

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Figure 3. Induction of cell apoptosis in the synovium by intraarticular injection of double-stranded microRNA-15a (miR-15a), as determined by immunohistochemical analysis of caspase 3 expression, indicating the number of apoptotic cells. On day 3 after intraarticular injection of the small interfering RNA–atelocollagen complex (control) (a), there was only slight or no expression of caspase 3, whereas on day 3 after intraarticular injection of double-stranded miR-15a–atelocollagen complex (b), the expression of caspase 3 was increased, as indicated by caspase 3 staining (left) and the merged image of caspase 3 plus 4′,6-diamidino-2-phenylindole (DAPI) (right). Bars = 100 μm.

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Distribution of injected double-stranded miR-15a in other organs.

To investigate whether intraarticular injection of double-stranded miRNA-15a–atelocollagen complex into the knee joint would affect other tissues, the levels of miR-15a expression in the internal organs (liver, lung, heart, kidney, and spleen) were determined by quantitative RT-PCR. We observed no significant difference in the expression of miR-15a in the lung, spleen, kidney, and heart of mice in the experimental group and mice in the control group. However, the expression level of miRNA-15a in the liver was significantly higher in the experimental group than in the control group (Figure 4).

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Figure 4. Distribution of injected double-stranded microRNA-15a (miR-15a) in the joints and other organs at 24 hours following injection, as determined by quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis. Expression of miR-15a after injection of the double-stranded miR-15a–atelocollagen complex was significantly higher in the knee joint and liver than the expression following injection of the small interfering RNA–atelocollagen complex (control). Values are the mean and SD of 6 mice per group. = P < 0.05. NS = not significant.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Recently, miRNA have attracted attention because there is evidence of miRNA functioning in human diseases; therefore, miRNA might be a novel therapeutic target for human diseases. For example, the expression of let-7 has been shown to be lower in cancerous lung tissue than in normal lung tissue, resulting in high levels of expression of the Ras gene (20). MicroRNA-146 is intensely expressed in RA synovial tissue (11). Therefore, regulation of the endogenous miRNA that are associated with human diseases could open the door to a new therapeutic strategy. Therapeutic trials aimed at silencing miRNA in vivo have been described (13, 14). Tazawa et al (15) reported that local injection of double-stranded miR-34a complexed with atelocollagen could suppress tumor growth in mice.

In the present study, we demonstrated the successful transfection of double-stranded miR-15a complexed with atelocollagen into synovial cells in the knee joints of arthritic mice. Atelocollagen has been reported to be a carrier biomaterial for gene delivery both in vitro and in vivo. Atelocollagen-mediated siRNA or miRNA delivery has been reported to be effective in gene silencing following local injection directly into tumors and to be effective in treating bone-metastatic tumors following intravenous injection because atelocollagen complexed with siRNA is resistant to nuclease and can be efficiently transduced into cells (15, 21). Schiffelers et al (22) reported that local electroporation of siRNA for tumor necrosis factor α in joint tissues could inhibit collagen-induced arthritis in mice by local interference with RNA. In the current study, we injected atelocollagen-mediated double-stranded miRNA into the knee joint because intraarticular injection is a method that is both simple and commonly used in the clinical setting. We found that synthetic double-stranded miRNA complexed with atelocollagen was efficiently taken up by cells and was functional in cells after intraarticular injection without electroporation. This technique may therefore serve as a useful tool for studying the function of miRNA through the overexpression of specific miRNA in arthritic disease.

In contrast, after injection of the double-stranded miR-15a–atelocollagen complex, miR-15a uptake by the liver was also noted, whereas the lung, spleen, kidney, and heart showed no significant uptake. The uptake of miR-15a in these internal organs in the experimental group was not as high as that in the knee joint. In addition, there was no difference in the expression of caspase 3 in the liver of mice in the experimental group and the control group, as determined by immunohistochemistry (data not shown). However, a part of the injected double-stranded miRNA dose may have moved from the joint cavity into the systemic circulation. These results must be addressed before clinical studies using systemic miRNA can be undertaken.

It has been reported that the function of miR-15a is in the down-regulation of Bcl-2 protein expression and the promotion of cell apoptosis without affecting the stability of messenger RNA (16). Bcl-2 is a central player in the genetic program of eukaryotic cells, favoring survival by inhibiting cell death (23). Bcl-2 is essential to the processes of apoptosis because it suppresses the initiation of the cell death process. In addition, overexpression of Bcl-2 protein has been reported in many types of human cancers, including leukemia, lymphoma, and carcinoma (24). The apoptotic process in RA synovial fibroblasts might be suppressed by the overexpression of Bcl-2. In the current study, miR-15a was down-regulated in arthritic mouse synovium and a high level of Bcl-2 expression was observed.

To determine whether an immune response to miRNA does or does not induce apoptosis, we used miR-124 (a neuron-specific miRNA) as the control miRNA. Cell apoptosis was not observed in arthritic synovium following the intraarticular injection of miR-124 (data not shown). Therefore, no innate immune response to miRNA, which then induced cell apoptosis, was detected.

This trial successfully induced cell apoptosis by the intraarticular injection of double-stranded miR-15a and inhibited the translation of Bcl-2 protein in arthritic synovium. The limitation of this study is that it was not possible to demonstrate a complete therapeutic effect, such as the remission of arthritis. Because the pathogenesis of arthritis is quite complicated, it would be impossible to resolve the condition simply by inducing cell apoptosis via inhibition of the Bcl-2 cascade. It would require a cocktail of several double-stranded miRNA or antisense oligoribonucleotides that would regulate inflammation, cell proliferation, etc. The current study showed the possibility of a new strategy for inducing the overexpression of miRNA in vivo by the intraarticular injection of double-stranded miRNA into the joint. Further study is needed, however, before clinical studies can be begun.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nakasa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Nagata, Nakasa, Ochi.

Acquisition of data. Nagata, Nakasa, Ishikawa, Shibuya, Yamasaki.

Analysis and interpretation of data. Nagata, Nakasa, Mochizuki, Miyaki, Adachi, Asahara.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The authors thank Professor Michihiro Hide for permission to use the instruments (the NanoDrop 1000 [Thermo Scientific, Tokyo, Japan] for protein density measurements and the LAS-1000 Plus Luminescent Image Analyzer [Fujifilm, Tokyo, Japan] for analysis of chemiluminescence) and Ms Kaori Ishii for technical support for the Western blot studies (Department of Dermatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES