Bruno Augusto Linhares Almeida Mariz

DNA methylation analysis of cancer-related genes in oral epithelial cells of healthy smokers

AIM The aim of this study was to investigate the smoking habit influence on DNA methylation status in the promoters of the cancer related-genes MLH1, hTERT and TP53 in oral epithelial cells of healthy subjects. MATERIALS AND METHODS DNA methylation analysis was performed using methylation-sensitive restriction enzymes (MSRE) in oral epithelial cells from non-smokers, smokers and ex-smokers. RESULTS The investigated CpG dinucleotides located at HhaI and HpaII sites in the MLH1 gene promoter were observed to be fully methylated in the majority of DNA samples from the smoker group and statistical differences were found between non-smokers and smokers and between smokers and ex-smokers (p<0.05). The same was observed in the hTERT gene promoter at HhaI sites (p<0.05) and for HpaII sites the unmethylated condition was more frequent in smokers in comparison to non-smokers (p<0.05). For TP53, no differences were found among groups (p>0.05), with the fully methylated condition found to be a common event in healthy oral epithelial cells. CONCLUSION We conclude that smoking may induce changes in DNA methylation status in cancer-related genes of oral epithelial cells and that the cessation of smoking is capable of reversing this process. Based on our data, we suggest that DNA methylation status of the hTERT and MLH1 gene promoters are promising markers for screening a set of smoking-related alterations in oral cells.

Influence of smoking on methylation and hydroxymethylation levels in global DNA and specific sites of KRT14, KRT19, MIR-9-3 and MIR-137 genes of oral mucosa

OBJECTIVE The aim of this study was to investigate the influence of smoking on the methylation and hydroxymethylation status of global DNA and specific sites of KRT14, KRT19, miR-9-3 and miR-137 genes in the healthy oral mucosa. METHODS Samples of oral epithelial cells were collected using mouthwash from a population of former and current smokers and those who had never smoked. Genomic DNA was extracted, and global DNA methylation and hydroxymethylation was performed using an ELISA-based technique; DNA methylation at specific sites was performed using Methylation-Specific PCR (MSP) (KRT14, miR-9-3 and miR-137) or Methylation-Sensitive Restriction Enzymes (MSRE) (KRT19). K14 and K19 protein expression was analysed by immunohistochemistry. RESULTS Higher levels of global DNA methylation were found in current smokers with over 15 years of consumption (p=0.04), but no differences were found in relation to global DNA hydroxymethylation. No differences in global DNA methylation and hydroxymethylation levels were found in relation to age or gender. Global DNA methylation was higher than the hydroxymethylation level (p<0.001) but they were not correlated in the oral mucosa. For specific sites, miR-9-3 hypomethylation was detected in current smokers (p<0.001). Additional analysis showed no difference in the methylation status when age, gender, period of consumption or amount of cigarettes were considered for any of the studied genes. K19 expression was higher in current smokers in comparison to former smokers and those who had never smoked (p<0.05). CONCLUSION We concluded that smoking habits were capable of inducing changes in global DNA methylation, miR-9-3 methylation status and K19 expression.

MONOSTOTIC FIBROUS DYSPLASIA

A 63-year-old male was referred to the OROCENTRO Clinic Piracicaba Dental School due to a swelling at the right side of the face. Patient stated that first noted the swelling for over 20 years. The patient was submitted to multiple tooth extractions on the region due to pain. After extractions pain ceased, nevertheless the swelling kept growing. Extraoral examination revealed a hard swelling on the right side of the face causing elevation of the nose wing and deletion of nasolabial groove. Intraoral examination revealed a hard, normochromic swelling on the right maxilla affecting both palatine and vestibular surfaces with deletion of maxillary vestibule. Digital panoramic radiography showed a poorly defined radiopaque image with “ground glass” aspect. Computed tomography revealed a mixed density image affecting the floor of the maxillary sinus and vestibular and palatine cortical bone of the right maxilla. Incisional biopsy was performed under local anaesthesia on the vestibular surface of the right maxilla using a Trephina drill (7 mm X 180mm). Histopathological analysis presented fine branching curvilinear/irregular trabeculae of woven bone among a fibrous cellular stroma and was compatible with Monostotic Fibrous Dysplasia. Patient did not present signs of post-operatory infection or dehiscence and was referred to an appropriate treatment service for surgical planning.