Asghar Ghasemi

MicroRNA-219-1 (miR-219-1) acts as a tumor suppressor in a variety of cancers but, the regulatory epigenetic mechanism involved in its gene expression level has not been studied. Using real-time polymerase chain reaction (real-time PCR) and bisulfite genomic sequencing technology, promoter methylation level of miR-219-1 and gene expression levels of miR-219-5p and miR-219-1-3p were determined respectively, in glioblastoma multiforme (GBM) (n = 31), their adjacent normal tissues (n = 31), and GBM U87 cell line. Following treatment of GBM U87 cells with 5-aza-2′-deoxycitidine (5-aza-dC), miR-219-1 promoter methylation, their target mRNA, and protein levels were determined by genomic bisulfite modification, real-time-PCR, and ELISA techniques, respectively. Our results showed that gene expression levels of miR-219-5p and miR-219-1-3p were significantly lower in GBM patients relative to their adjacent normal tissues (p < 0.01). MiR-219-1 promoter had a high level of methylation in GBM tissues (p < 0.01) and a negative correlation was observed between the miRNAs gene expression and methylation levels in GBM tissues (p < 0.01). Treatment of GBM U87 cells by 5-aza-dC decreased the level of miR-219-1 methylation, amount of target mRNA, and levels of cyclin A2 and mucin 4 (MUC4) proteins, and increased the expression levels of miR-219-5p and miR-219-1-3p (p < 0.01). Using external miR-219-5p and miR-219-1-3p, the expression of cyclin A2 and MUC4 were suppressed and proliferative activity of the U87MG cell line was reduced (p < 0.01). These findings suggested that DNA methylation has a crucial role in the regulation of miR-219-1 gene expression and that hypermethylated miR-219-1 may be involved in GBM pathogenesis.

Deregulated expressions of tumor-suppressive microRNAs (miRNAs) by epigenetic aberrations has a critical role in tumorigenesis. The aim of the present study was to investigate the epigenetic aberrations of miR205 and to understand how this modification may contribute to molecular events in glioblastoma multiform (GBM). Quantitative RT-PCR and bisulfite genomic sequencing techniques were used to investigate gene expression and methylation levels of miR-205 in GBM tissues (n = 23), their matched adjacent normal tissues (n = 23) and glioblastoma U87MG cell line. Following treatment of cells with 5-aza-2'-deoxycitidine (5-aza-dC), DNA methylation and gene expression levels of miR-205 gene and protein expressions of its target mRNA were investigated. Our study showed that gene expression level of miR-205 decreased in GBM tissues compared to controls (p < 0.01) and lower expression was significantly correlated with this miRNA promoter hypermethylation (r = -78; p < 0.01). Cell treatment with 5-aza-dC restored the hypermethylated promoter and gene expression of the miR205 and decreased target mRNA and proteins levels (p < 0.01). In summary, our results offered that miR-205 is an epigenetically silenced tumor suppressive miRNA in GBM, suppresses enhanced target mRNA when induced by DNA demethylating agents.

Next-generation sequencing (NGS) is the catch all terms that used to explain several different modern sequencing technologies which let us to sequence nucleic acids much more rapidly and cheaply than the formerly used Sanger sequencing, and as such have revolutionized the study of molecular biology and genomics with excellent resolution and accuracy. Over the past years, many academic companies and institutions have continued technological advances to expand NGS applications from research to the clinic. In this review, the performance and technical features of current NGS platforms were described. Furthermore, advances in the applying of NGS technologies towards the progress of clinical molecular diagnostics were emphasized. General advantages and disadvantages of each sequencing system are summarized and compared to guide the selection of NGS platforms for specific research aims.

Aberrant DNA methylation has been shown to inactivate tumor suppressor genes during carcinogenesis. MicroRNA-149 (miR-149) was recently demonstrated to function as a tumor suppressor gene in glioblastoma multiforme (GBM). However, the potential linkage of miR-149 levels and the underlying epigenetic regulatory mechanism in human GBM has not been studied. We used quantitative real-time polymerase chain reaction to investigate the levels of miR-149 in GBM tissues, their matched adjacent normal tissues, and glioblastoma U87MG cell line. Using bisulfite genomic sequencing technology, DNA methylation status of upstream region of miR-149 was evaluated in study population groups and the U87MG cell line. After treatment of cells with 5-aza-2′-deoxycitidine (5-aza-dC), the DNA methylation status, gene expression, and target protein levels of miR-149 were investigated. Our studies revealed that methylation and expression levels of miR-149 were significantly increased and decreased, respectively in GBM patients relative to the adjacent normal tissues (P < 0.01). MiR-149 suppressed the expression of AKT1 and cyclin D1 and reduced the proliferative activities of the U87MG cell line. Treatment of U87MG cells with 5-aza-dC reversed the hypermethylation status of miR-149, enhanced the expression of its gene, and decreased target mRNA and proteins levels (P < 0.01). These findings suggest that the methylation mechanism is associated with decreased expression levels of miR-149, which may in turn lead to the increased levels of its oncogenic target proteins.

Aberrant DNA hypermethylation contributes to many cancers by silencing structurally normal tumor suppressive genes. MicroRNA-153 (miR-153) exerts a tumor suppressive function in glioblastoma multiforme (GBM) by silencing oncogenic targets. However, the mechanism underlying miR-153 regulation in glioma cells has not been studied. The expression levels of miR-153 were determined by real-time PCR and genomic bisulfite modification technique was used to detect the DNA methylation status in the upstream region of miR-153 in GBM, their matched normal adjacent tissues, and the glioblastoma U87 cell line. Following treatment of cells with 5-aza-2'-deoxycitidine (5-aza-dC), the DNA methylation, gene expression and target proteins levels of miR-153 were determined. This study confirmed that miR-153 is significantly downregulated and hypermethylated in GBM tissues compared to their matched normal adjacent tissues. Increased methylation level of miR-153 was significantly correlated with reduced miR-153 expression in GBM tissue specimens. Demethylation of cells by 5-aza-dC treatment led to reduction of miR-153 methylation level, re-expression of candidate microRNA, and downregulation of its target proteins levels. Our data indicated that miR-153 acts as a tumor suppressor in GBM and is down-regulated by DNA methylation, suggesting that miR-153 may serve as a potential diagnostic or therapeutic target of GBM. (Clin. Lab. 2016;62:573-580. DOI: 10.7754/Clin.Lab.2015.150738)

Anadolu Kardiyoloji Dergisi, Anadolu Kardiyol Derg, The Anatolian Journal of Cardiology, Anatol J Cardiol, Cardiology, Cardiovascular Medicine, Surgery, Pediatric Cardiology, Angiology

In a recent study, a genome-wide scan has identified C771G (His241Gln) polymorphism of MLX interacting protein like (MLXIPL) gene that is associated with the level of plasma triglycerides. Since, no study has been reported on the association between MLXIPL gene and non-alcoholic fatty liver disease (NAFLD), we aimed to identify a connection between this genetic variation and NAFLD. Two hundred and thirteen patients with NAFLD and 252 healthy controls were entered into this study. MLXIPL genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Our study showed that the single nucleotide polymorphism (SNP) of MLXIPL is significantly associated with NAFLD. Significant differences between cases and controls were observed for MLXIPL genotype frequencies (p<0.002). The frequency of C allele of MLXIPL in patient group was higher than the control group (68.30% vs. 51.59%, respectively; p<0.05). C771G polymorphism in the MLXIPL gene potentially plays a significant role in pathophysiology of non-alcoholic fatty liver disease. Further research is needed to confirm this finding.

There is evidence showing the association between connexin 37 (Cx37) C1019T polymorphism and acute myocardial infarction (AMI). Since there has been no study that takes the polymorphisms of Cx37 and connexin 40 (Cx40) into consideration at the same time, we investigated the association between AMI and the polymorphism gene of two gap junction proteins Cx 37 and Cx40 which are important in the electrical coupling between arterial myocytes. 200 patients with acute myocardial infarction (AMI) and 185 healthy controls were included in this study. Cx37 and Cx40 genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). We identified that single nucleotide polymorphism (SNP) of Cx37 was significantly associated with AMI disease. Significant differences between cases and controls were observed for Cx37 genotype frequencies (p < 0.001, Chi2 = 16.528). The T allele of Cx37 had more frequency in the AMI group compared to the control group. (53.75% vs. 40%; p < 0.05). Subsequent analysis identified that, in contrast to the previous studies, there is a significant difference in women (p < 0.01) but not in men. We also found that the SNP of Cx40 was not significantly associated with AMI disease (p > 0.05). Our study showed that the -44A allele and -44AA genotype were not significantly different in the AMI and control groups (p > 0.05). It is suggested that the polymorphism in the Cx37 gene (but not Cx40 gene) potentially plays a significant role in the manifestation of AMI disease in Iranian population.