DNA methylation polymerase chain reaction (PCR) array of apoptosis-related genes in pleomorphic adenomas of the salivary glands.

OBJECTIVE: The aim of this study was to evaluate the DNA methylation profile in 22 apoptosis-related genes in pleomorphic adenomas (PAs) of the salivary glands, in comparison with normal salivary glands (NSGs), and to address the differences in methylation patterns between smaller and larger tumors. Additionally, we investigated if the hypermethylation of differentially methylated genes between NSGs and PAs impacted the messenger RNA (mRNA) transcription. DESIGN: Twenty-three fresh PA samples and 12 NSG samples were included. The PA samples were divided into 2 groups: PAs with clinical size larger than 2 cm (n = 12) and PAs with clinical size 2 cm or smaller (n = 11). DNA methylation at the promoter region of a panel of 22 genes involved in apoptosis was profiled by using a human apoptosis DNA methylation polymerase chain reaction array, and the transcriptional levels of genes showing differential methylation profiles between PAs and NSGs were assessed. RESULTS: TNFRSF25 and BCL2 L11 were highly methylated in PAs, in comparison with NSGs, irrespective of tumor size. However, no difference could be observed in the mRNA transcription between PAs and NSGs. CONCLUSIONS: Hypermethylation of the proapoptotic genes BCL2 L11 and TNFRSF25 is observed in PA. However, this phenomenon did not impact mRNA transcription.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

INTRODUCTION: Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. METHODS: Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. RESULTS: All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. CONCLUSIONS: The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions.

FOXP3 DNA methylation levels as a potential biomarker in the development of periapical lesions.

INTRODUCTION: Epigenetic mechanisms, such as DNA methylation, can modify gene expression patterns without changing the DNA sequence, comprising a tool that cells use to lock genes in the "off" position. Variations in the methylation profile have been correlated to a variety of human diseases. Here, we hypothesize that DNA methylation in immune response-related genes may contribute to the development of periapical lesions. METHODS: The DNA methylation patterns of 22 immune response-related gene promoters were evaluated in 137 human periapical granulomas, 8 apical cysts, and 31 healthy gingival tissues from 2 independent cohorts using a pathway-specific real-time polymerase chain reaction array (EpiTect Methyl II; Qiagen Inc, Valencia, CA). Messenger RNA expression analysis by qualitative polymerase chain reaction was also performed. SABiosciences's hierarchical clustering and methylation (Qiagen, Valencia, CA) and Prism6 software (GraphPad Software, Inc, La Jolla, CA) were used for data analysis. RESULTS: FOXP3 gene promoter showed the highest level of methylation in both periapical granulomas and apical cysts (P < .001), and methylation levels were inversely correlated with FOXP3 messenger RNA expression in the lesions. Furthermore, FOXP3 expression was prevalent in inactive lesions and was positively correlated with interleukin-10 and transforming growth factor beta levels. CONCLUSIONS: Our results suggest that FOXP3 acts as a master switch governing the development and function of T-regulatory cells, whose functions include the inhibition of immune responses and temper inflammation. The observed differential methylation patterns of FOXP3 in periapical lesions may be crucial in determining its suppressive activity and may be involved in periapical lesion development.

Relationship between microRNA expression levels and histopathological features of dysplasia in oral leukoplakia.

BACKGROUND: Increased expression of microRNAs (miRNAs), miR-21, miR-345, and miR-181b has been demonstrated in oral leukoplakia (OL) that progresses to oral squamous cell carcinoma (OSCC), suggesting a miRNA signature with potential prognostic value. On the basis of these findings, this pilot study aimed to investigate the cytological and histopathological features that are used to grade oral dysplasia and determine associations with the expression of these 3 potentially cancer-related miRNAs. We also compared the expression levels of these miRNAs in OL with normal oral mucosa and OSCC. METHODS: We evaluated miRNA expression by qPCR in 22 samples of OL demonstrating different grades of dysplasia, as well as 17 cases of OSCC, and 6 samples of normal oral mucosa. We associated the miRNAs expression profiles with cytological and histopathological features of OL. RESULTS: OSCC cases showed increased expression of all 3 miRNAs when compared with OL and normal oral mucosa. Increased expression of miR-21 was also observed in OL when compared with normal oral mucosa. We found a higher expression of miR-21 and miR-181b in OL that presented with an increased number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromasia. Increased expression of miR-21 was also detected in OL with abnormally superficial mitosis. Higher expression of miR-345 was observed in OL with an increased number and size of nucleoli or increased nuclear/cytoplasmic ratio. CONCLUSIONS: In conclusion, the present study shows that some cytological and histopathological parameters used to grade dysplasia are associated with altered miRNA expression.

Assessing the contribution of HRPT2 to the pathogenesis of jaw fibrous dysplasia, ossifying fibroma, and osteosarcoma.

OBJECTIVE: To investigate HRPT2 in jaw ossifying fibroma (OF), fibrous dysplasia (FD), and osteosarcoma (OS). STUDY DESIGN: We combined microsatellite loss of heterozygosity (LOH), HRPT2 sequence alterations at the mRNA level by reverse-transcription polymerase chain reaction (PCR), cDNA sequencing, and quantitative PCR (qPCR) and immunohistochemistry (IHC) in a total of 19 OF, 15 FD, and 9 OS. Because HRPT2 (parafibromin) interacts with cyclin D1, we investigated cyclin D1 expression with the use of qPCR and IHC. RESULTS: LOH was detected in 3/5 FD, 6/9 OF, and 2/2 OS heterozygous samples. LOH was not associated with decreased mRNA levels or HRPT2 protein expression except for 1 OF which harbored an inactivating mutation. However, this tumor did not display altered transcription or protein levels of HRPT2 nor cyclin compared with the other OF. CONCLUSIONS: The contribution of HRPT2 inactivation to the pathogenesis of OF, FD, and OS is marginal at best and may be limited to progression rather than tumor initiation.

Methylation pattern of IFNG in periapical granulomas and radicular cysts.

INTRODUCTION: Interferon-γ plays an important role in the pathogenesis of periapical lesions, and the methylation of IFNG has been associated with transcriptional inactivation. The purpose of the present study was to investigate IFNG promoter methylation in association with gene transcription and protein levels in periapical granulomas and radicular cysts. METHODS: Methylation-specific polymerase chain reaction was used to assess the DNA methylation pattern of the IFNG gene in 16 periapical granulomas and 13 radicular cyst samples. The transcription levels of IFNG mRNA were verified by quantitative real-time polymerase chain reaction, and protein expression was evaluated by immunohistochemistry. RESULTS: All the periapical lesion samples exhibited partial or total methylation of the IFNG gene. In addition, an increased methylation profile was found in radicular cysts compared with periapical granulomas. Increased IFNG mRNA expression was observed in the partially methylated periapical lesion samples relative to the samples that were completely methylated. CONCLUSIONS: The present study provides the first evidence of the possible impact of IFNG methylation on IFNG transcription in periapical lesions.