RESUMO
Eosinophilic colitis (EC) is a common cause of haematochezia in infants and young children. The exact pathomechanism is not understood, and the diagnosis is challenging. The role of microRNAs as key class of regulators of mRNA expression and translation in patients with EC has not been explored. Therefore, the aim of the present study was to explore the miRNA profile in EC with respect to eosinophilic inflammation. Patients enrolled in the study (n = 10) had persistent rectal bleeding, and did not respond to elimination dietary treatment. High-throughput microRNA sequencing was carried out on colonic biopsy specimens of children with EC (EC: n = 4) and controls (C: n = 4) as a preliminary screening of the miRNA profile. Based on the next-generation sequencing (NGS) results and literature data, a potentially relevant panel of miRNAs were selected for further measurements by real-time reverse transcription (RT)-PCR (EC: n = 14, C: n = 10). Validation by RT-PCR resulted in significantly altered expression of miR-21, -31, -99b, -125a, -146a, -184, -221, -223, and -559 compared to controls (p ≤ 0.05). Elevation in miR-21, -99b, -146a, -221, and -223 showed statistically significant correlation to the extent of tissue eosinophilia. Based on our results, we conclude that the dysregulated miRNAs have a potential role in the regulation of apoptosis by targeting Protein kinase B/Mechanistic target of rapamycin (AKT/mTOR)-related pathways in inflammation by modulating Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related signalling and eosinophil cell recruitment and activation, mainly by regulating the expression of the chemoattractant eotaxin and the adhesion molecule CD44. Our results could serve as a basis for further extended research exploring the pathomechanism of EC.
Assuntos
Colite/metabolismo , Colo/metabolismo , Eosinofilia/metabolismo , MicroRNAs/genética , Apoptose , Criança , Pré-Escolar , Colite/genética , Colite/patologia , Colo/patologia , Eosinofilia/genética , Eosinofilia/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , TranscriptomaRESUMO
AIM: To evaluate the role of microRNA (miR)-146a, -155 and -122 in the duodenal mucosa of pediatric patients with Crohn's disease (CD) and the effect of transforming growth factor-ß (TGF-ß) on these miRs in duodenal epithelial and fibroblast cells. METHODS: Formalin-fixed, paraffin-embedded biopsies derived from the macroscopically inflamed (CD inflamed: n = 10) and intact (CD intact: n = 10) duodenal mucosa of pediatric CD patients and control children (C: n = 10) were examined. Expression of miR-146a, -155 and -122 was determined by real-time polymerase-chain reaction (PCR). The expression of the above miRs was investigated in recombinant human TGF-ß (1 nmol/L, 24 h) or vehicle treated small intestinal epithelial cells (CCL-241) and primary duodenal fibroblast cells derived from healthy children as well. RESULTS: Expression of miR-146a was significantly higher in the inflamed duodenal mucosa compared to the intact duodenal mucosa of children with CD (CD inflamed: 3.21 ± 0.50 vs CD intact: 0.62 ± 0.26, P ≤ 0.01) and to the control group (CD inflamed: 3.21 ± 0.50 vs C: 1.00 ± 0.33, P ≤ 0.05). The expression of miR-155 was significantly increased in the inflamed region of the duodenum compared to the control group (CD inflamed: 4.87 ± 1.02 vs CONTROL: 1.00 ± 0.40, P ≤ 0.001). The expression of miR-122 was unchanged in the inflamed or intact mucosa of CD patients compared to controls. TGF-ß treatment significantly decreased the expression of miR-155 in small intestinal epithelial cells (TGF-ß: 0.7 ± 0.083 vs CONTROL: 1 ± 0.09, P ≤ 0.05) and also the expression of miR-146a (TGF-ß: 0.67 ± 0.04 vs CONTROL: 1 ± 0.15, P ≤ 0.01) and miR-155 (TGF-ß: 0.72 ± 0.09 vs CONTROL: 1 ± 0.06, P ≤ 0.05) in primary duodenal fibroblasts compared to corresponding vehicle treated controls. TGF-ß treatment did not influence the expression of miR-122. CONCLUSION: The elevated expression of miR-146a and -155 in the inflamed duodenal mucosa of CD patients suggests the role of these miRs in the pathomechanism of inflammatory bowel disease. Anti-inflammatory TGF-ß plays an important role in the regulation of the expression of these miRs.
Assuntos
Doença de Crohn/genética , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/genética , Adolescente , Criança , Doença de Crohn/metabolismo , Duodeno/citologia , Duodeno/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/farmacologiaRESUMO
AIMS: In polycystic ovary syndrome (PCOS), metabolic and cardiovascular dysfunction is related to hyperandrogenic status and insulin resistance, however, Vitamin D3 has a beneficial effect partly due to its anti-oxidant capacity. Nitrative stress is a major factor in the development of cardiovascular dysfunction and insulin resistance in various diseases. Our aim was to determine the effects of vitamin D3 in a rat model of PCOS, particularly the pathogenic role of nitrative stress. MAIN METHODS: Female Wistar rats weighing 100-140g were administered vehicle (C), dihydrotestosterone (DHT) or dihydrotestosterone plus vitamin D3 (DHT+D) (n=10 per group). On the 10th week, acetylcholine (Ach) induced relaxation ability of the isolated thoracic aorta rings was determined. In order to examine the possible role of endothelial nitric oxide synthase (eNOS) and cyclooxygenase-2 (COX-2) pathways in the impaired endothelial function, immunohistochemical labeling of aortas with anti-eNOS and anti-COX-2 antibodies was performed. Leukocyte smears, aorta and ovary tissue sections were also immunostained with anti-nitrotyrosine antibody to determine nitrative stress. KEY FINDINGS: Relaxation ability of aorta was reduced in group DHT, and vitamin D3 partly restored Ach induced relaxation. eNOS labeling was significantly lower in DHT rats compared to the other two groups, however COX-2 staining showed an increment. Nitrative stress showed a significant increase in response to dihydrotestosterone, while vitamin D3 treatment, in case of the ovaries, was able to reverse this effect. SIGNIFICANCE: Nitrative stress may play a role in the pathogenesis of PCOS and in the development of the therapeutic effect of vitamin D3.