Methylation status of interleukin-6 gene promoter in patients with Behçet's disease / Estatus de la metilación del promotor del gen interleucina-6 en pacientes con síndrome de Behçet

BACKGROUND: IL-6 mRNA expression is significantly high in many autoimmune diseases such as Behçet's disease; this is often related with more aggressive phenotypes. Nevertheless, the essential molecular process for its high expression has not been completely realized. The aim of this study was undertaken to estimate the gene copy number variation and promoter methylation to IL-6's high expression. METHODS: This study was performed on 51 patients and 61 healthy controls. Initially, DNA and RNA were extracted from all specimens. Promoter methylation levels of IL-6 were evaluated by MeDIP-qPCR technique. Also, IL-6 gene expression was measured by Real-time PCR. After that, we evaluated the relationship between gene expression and methylation, as well as their relationship with clinical specification. RESULTS: As we expected, the expression level of IL-6 gene increased significantly in the patient group compared to the healthy subjects. Also, the relative promoter methylation level of the IL-6 mRNA was significantly lower in patient group compared to healthy group (p < 0.001). DISCUSSION: We disclosed that the promoter hypomethylation may be considered as one of the main defects for IL-6 mRNA high expression in patients with Behçet's disease

ANTECEDENTES: La expresión de ARNm de IL-6 es significativamente elevada en muchas enfermedades autoinmunes, tales como el síndrome de Behçet, y ello se relaciona a menudo con fenotipos más agresivos. Sin embargo, no se ha comprendido plenamente el proceso molecular esencial para esta expresión elevada. El objetivo de este estudio fue la estimación de la variación del número de copias del gen, y la metilación del promotor de la expresión elevada de IL-6. MÉTODOS: Este estudio se realizó en 51 pacientes y 61 controles sanos. Al inicio, se extrajo ADN y ARN de todas las muestras. Se evaluaron los niveles de metilación del promotor de IL-6 mediante la técnica MeDIP-qPCR. También se midió la expresión del gen IL-6 mediante PCR a tiempo real. Tras ello, evaluamos la relación entre la expresión del gen y la metilación, así como su relación con la especificación clínica. RESULTADOS: Según lo previsto, el nivel de expresión del gen IL-6 se incrementó significativamente en el grupo de pacientes, con respecto a los sujetos sanos. También encontramos que el nivel relativo de metilación del promotor de ARNm de IL-6 fue considerablemente menor en el grupo de pacientes, con respecto al grupo sano (p < 0,001). DISCUSIÓN: Concluimos que la hipometilación del promotor puede considerarse uno de los defectos principales de la expresión elevada de ARNm de IL-6, en los pacientes con síndrome de Behçet

Methylation status of interleukin-6 gene promoter in patients with Behçet's disease.

BACKGROUND: IL-6 mRNA expression is significantly high in many autoimmune diseases such as Behçet's disease; this is often related with more aggressive phenotypes. Nevertheless, the essential molecular process for its high expression has not been completely realized. The aim of this study was undertaken to estimate the gene copy number variation and promoter methylation to IL-6's high expression. METHODS: This study was performed on 51 patients and 61 healthy controls. Initially, DNA and RNA were extracted from all specimens. Promoter methylation levels of IL-6 were evaluated by MeDIP-qPCR technique. Also, IL-6 gene expression was measured by Real-time PCR. After that, we evaluated the relationship between gene expression and methylation, as well as their relationship with clinical specification. RESULTS: As we expected, the expression level of IL-6 gene increased significantly in the patient group compared to the healthy subjects. Also, the relative promoter methylation level of the IL-6 mRNA was significantly lower in patient group compared to healthy group (p<0.001). DISCUSSION: We disclosed that the promoter hypomethylation may be considered as one of the main defects for IL-6 mRNA high expression in patients with Behçet's disease.

Angiopoietin-like protein 8 (betatrophin) may inhibit hepatocellular carcinoma through suppressing of the Wnt signaling pathway.

OBJECTIVES: Hepatocellular carcinoma (HCC) is one of the leading fatal neoplasms and the most common primary liver malignancy worldwide. Peptide hormone ANGPTL8 (betatrophin) may act as an important regulator in HCC development through the Wnt/ß-catenin pathway. We aimed to evaluate the effects of recombinant ANGPTL8 on Wnt/ß-catenin signaling in human liver carcinoma cells (HepG2) and their viability. MATERIALS AND METHODS: The expression of ANGPTL8 was conducted in the pET-21b-E. coli Bl21 (DE3) system and the produced peptide was purified. HepG2 cells were treated with different concentrations of ANGPTL8 (25, 50, 100, 150, 200, and 250 ng/ml) for 24, 48, and 72 hr. MTT assay was performed to detect the viability of treated cells, and apoptotic induction by ANGPTL8 was measured by flow cytometry assay. Finally, using qRT-PCR the mRNA levels of Wnt signaling modulators WIF-1 and ß-catenin were evaluated in treated cells. RESULTS: MTT assay showed that ANGPTL8 inhibits proliferation of HepG2 cells moderately in a time-independent manner. The highest concentration of the ANGPTL8, 250 ng/ml, reduced cell proliferation after 24, 48, and 72 hr similarly about 30%. In the same concentration of ANGPTL8, after 24 hr of treatment flow cytometry assay revealed a mild increase in early and late apoptosis up to 7.7 and 14.3%, respectively. The qRT-PCR showed that in a concentration-dependent and time-independent fashion, the expression of WIF-1 and ß-catenin genes respectively increased and decreased significantly (P<0.05). CONCLUSION: Our findings suggest that ANGPTL8 may act as a moderate suppressor against HCC cell proliferation possibly via affecting Wnt signaling modulators.

Analysis of Methylation and Expression Profile of Foxp3 Gene in Patients with Behçet's Syndrome.

Forkhead box P3 (Foxp3) gene is an important means in the Treg cells function, in both maintenances of immune tolerance and regulation of response. Epigenetic modifications of the foxp3 gene at its regulatory regions control the chromatin accessibility for the transcription factors and other transcriptional regulators in order to control Foxp3 expression. In addition, the methylation status of CpG islands within the Foxp3 promoter and regulatory elements regulate the expression of Foxp3. This study was performed to assess the role of the foxp3 gene in patients with Behçet's syndrome (BS). Venous blood samples were collected from all participants and peripheral blood mononuclear cells (PBMC) were extracted through Ficoll-Hypaque method. Genomic DNA was randomly sheared by sonication and immunoprecipitated with a monoclonal antibody. The status methylation of the foxp3 gene was estimated in 108 blood samples of active BS patients and healthy individuals (controls); using methylation DNA immunoprecipitation (MeDIP) technique. Expression analysis was carried out; using Real-time PCR. The expression of foxp3 gene in the patients' group (mean±SD: 1.79±1.12) was significantly lower than the healthy group (mean±SD: 2.73±1.33) (p<001). Also, the methylation levels of Foxp3 promoter showed that its level in patients (mean±SD: 2.3±1.16) was higher than the healthy group (mean±SD: 1.85±0.59). However, this increase was not statistically significant (p>0.05). Also, these results indicated that increasing the amount of methylation of the foxp3 gene by reducing its expression leads to an increase and intensifying of the disease. The decrease in Foxp3 expression is possibly associated with hypermethylation of the gene, and it can be considered as a risk factor for BS. Future studies may be needed to identify the capability of specific DNA methylation alterations in this syndrome.

Emergency separation of craniopagus twins: case report.

Craniopagus is a very rare congenital anomaly that tends to affect females more often than males. It is classified as partial or total. Most affected twins are either stillborn or die during the perinatal period. Those who survive birth should undergo detailed radiological evaluations soon after their condition becomes stable so that the precise anatomy of the conjoined part can be defined and surgery can be planned in detail by a multidisciplinary team. Recommendations for decreasing the risk of unsuccessful surgery include performing surgery on an elective basis after extensive preoperative radiological evaluations as well as when the twins are at an acceptable age and weight for a complex surgical separation, generally as staged procedures. In addition, the operation should be performed by a well-equipped expert multidisciplinary team. When one of the conjoined twins dies, however, surgical separation cannot be postponed because the shared circulatory system predisposes the alive child to life-threatening complications, including coagulopathy. The authors report on the successful separation of craniopagus twins performed on an emergency basis at 32 weeks of gestational age because of the sudden death of one of them. At the time of separation surgery, the twins each weighed 1250 g. To the best of the authors' knowledge, this is the youngest age and lowest weight yet reported for successful surgical separation. The surviving twin developed a pseudomeningocele, which required a second operation and placement of a cystoperitoneal shunt 4 months after the operation. Additional surgery is planned to repair a cranial defect that resulted from the pseudomeningocele, but his general physical and mental condition was otherwise good at latest follow-up (12 months after separation surgery).