miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.


Introduction
Periodontitis is defined as a chronic, inflammatory, and multifactorial disease; it is associated with a dysbiotic biofilm and characterized by the progressive destruction of the dental insertion apparatus. 1 Epidemiological studies revealed that most adult individuals are affected by mild and moderate forms of this disease, while 5-20% of any population has severe periodontitis. In Brazil, the periodontitis prevalence is higher than the global average. 2 If not treated, periodontitis is a risk factor for prominent noncommunicable diseases, including pneumonia, gastritis, diabetes mellitus, and chronic renal disorders; also, the number of lost teeth is predictive of mortality by cardiovascular conditions. 3 As a multifactorial disease, genetic and (rs2424913), on the other hand, enhances the transcription of the sequence. 6 In fact, some studies demonstrated that rs1801133 is associated with global and site-specific hypomethylation, 7 while rs2424913 is associated with hypermethylation of specific genes. 8 DNA methylation profile alterations have been described in periodontitis, especially in Toll-like receptor, cytokine, and metalloproteinase genes. 9,10 Notably, some of these studies reported epigenetic alterations outside of gingival tissue, such as buccal epithelium and blood. This phenomenon may suggest these changes are not limited to the gingiva and, at the same time, reflect the cellular condition. Information on the methylation profile of microRNA genes (miR) and on the genes involved in the oxidative stress pathway are still scarce. However, the microRNA expression as well as antioxidant proteins, such as superoxide dismutase and catalase, are altered in periodontitis patients' gingival tissue cells and saliva. 11,12 It speculated that genetic polymorphisms in DNA methylation pathway genes (MTHFR and DNMT) and methylation profile alterations in microRNA genes and in genes from the oxidative stress pathway may be involved in periodontitis pathogenesis. This study focused on these genes because, as mentioned earlier, MTHFR and DNMT are involved in DNA methylation processes and both polymorphisms and methylation of these genes are involved in inflammatory diseases, [13][14][15] probably affecting the levels of products originated during the folate metabolic cycle, such as homocysteine and SAM radicals. 7 Genes related to oxidative stress pathways, such as SOD and CAT, have been associated with periodontitis, 12 while microRNA genes are involved in inflammatory diseases by silencing several mRNA targets related to cellular processes, including cellular homeostasis, proliferation, differentiation, development, growth, and apoptosis, 11 particularly the miR-9 family targets transcripts of inflammatory cytokines. 16,17 However, to the best of our knowledge, nothing is known, in the context of periodontitis, about genetic and epigenetic marks in the above genes. Therefore, the main objective of this study was to investigate the association between the MTHFR rs1801133 and the DNMT3B rs2424913 polymorphisms, and methylation profiles from MTHFR, miR-9-1, miR-9-3, superoxide dismutase (SOD1), and catalase (CAT) genes with periodontitis. The study also assessed the association between these The amount and the purity of the isolated DNA were measured with a Nanodrop spectrophotometer using the OD 260/280 ratio. The sample was considered pure when the mean value between two readings was 1.8 or greater.

Genetic polymorphism analysis of MTHFR and DNMT3B
The analysis of the single nucleotide polymorphisms

Statistical analysis
All data were compiled in an Excel ® spreadsheet.
Descriptive statistics was used for demographic data.
The Hardy-Weinberg equilibrium (HWE) was calculated for each polymorphism using the chi-squared test.
The Fisher's Exact or the chi-squared tests were also used for the analysis of possible associations between genotypic frequencies, allelic frequencies, methylation profiles, and the outcome (periodontitis), as well as the association between the methylation profiles and the presence of polymorphisms. All analyses were performed using the BioEstat 5.3 software. p-values <0.05 were considered statistically significant.

Results
Demographic data are shown in Table 1.

Methylation profile analysis
For MTHFR, the unmethylated profile appeared more frequently in both groups (92.5% in the control group and 90% in the periodontitis group).
For miR-9-1, methylation occurred in 100% of the periodontitis patients but only in 77.5% of the control group participants (p=0.0024). On the other hand, miR-9-3 showed a partially methylated profile in both groups (100%; Table 1 and Figure 2). For SOD1, the unmethylated profile was the most frequent in both groups (85% of the control group and 79% of the periodontitis group). For CAT, although no significant difference was observed between the two groups, there was a tendency towards methylation in the periodontitis group, since the methylated profile was more common amongst these patients (55%=50% partially methylated +5% methylated) when compared with the control group (42.5%=40% partially methylated +2.5% methylated; p=0.077; miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis J Appl Oral Sci. 2020;28:e20190583 5/11 Table 1 and Figure 3). The DNMT3B TT genotype and the miR-9-1 methylation profile were considered for statistical association analysis, because both genes showed a statistically significant difference between the two groups. However, no association between these variables and periodontitis was found.

Discussion
Periodontitis has been associated with genetic polymorphisms in several cellular pathways. 25  analyzed. This study focused on the genes that had  These data collectively demonstrate that this profile is common in buccal epithelial cells and is not associated with oral inflammation. The same CpG site analyzed herein has been studied by our group in patients with diabetic complications; the hypermethylated profile was detected in blood. 14 The data of this study concerning promoter methylation in microRNA genes revealed that the partially methylated miR-9-3 condition was the standard profile for buccal epithelial cells in individuals with or without periodontitis, while the miR-9-1 methylation was more common in periodontitis patients. According to definitions found in a microRNA database (http://www.mirdb.org), all three loci for miR-9 genes (1, 2 and 3) in the human genome can be transcribed into the same mature miR-9 sequence.
The miRDB database shows miR-9 may have 991 predictive targets. Hypermethylation of these genes and a decrease in their own expression may increase the levels of such targets, as has been recently shown by Marinho, et al. 32 (2019). In this study, levels of the rap guanine nucleotide exchange factor, a miR-9 target, were higher in the periodontitis patients' gingival crevicular fluid. The signaling cascade in which this factor participates may be involved in apoptosis, integrin-mediated signal transduction, and cellular transformation. Another target, the transforming growth factor β-1 (TGF-β1) receptor, is also expressed in the gingival tissue of individuals with periodontal disease. 16 It binds to the anti-inflammatory cytokine TGF-β1, which is involved in tissue regeneration and cellular processes such as proliferation, growth, and differentiation.
miR-9-1 hypermethylation has also been observed in cervical and ovarian cancer, and myeloma. 17,33,34 A study with HeLa cells demonstrated that miR-9 hypermethylation reduces its expression and is associated with enhanced interleukin 6 (IL-6) levels, a finding that may indicate that IL-6 transcripts are targets for miR-9 17 . Interleukin 6 levels are higher in  This study limitations consist of a low sample size for polymorphism studies due to a narrow sample collection period and to well-defined inclusion criteria, and for these reasons, data on the periodontitis group was not subgrouped by disease severity. However, the data of the study referred to the DNMT3B polymorphism rs2424913, which had not been previously studied in the periodontitis context. The finding of this study should be further explored in larger populations with different ethnic backgrounds before any conclusions can be drawn about the role of this gene in periodontitis. Although this study on DNA methylation did not involve the inflammation site, it still showed that profile alterations in buccal mucosa cells, especially in miR-9-1, may be involved in periodontitis.
As a multifactorial disease, periodontitis should be investigated focusing on an individual's genetic and epigenetic aspects. The latter can be modulated by individual habits and even be considered as therapeutic targets. Therefore, the data of this study contribute to the elucidation of molecular mechanisms involved in periodontitis development.
Aside from cytokine genes, Toll-like receptors, matrix metalloproteinases, and other molecules mentioned in the literature, it demonstrated that genes involved in DNA methylation and translation inhibition may be involved in periodontitis. Molecular data can be collectively tabulated into a panel of genes indicating susceptibility to periodontal disease and be used as a tool for personalized medicine, contributing to better diagnosis, prognosis, and treatment.
In summary, the conclusion is that the genotype TT DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis. The miR-9-1 promoter methylation yields a relative risk of 1.29 for periodontitis development, while a promoter region polymorphism in the DNMT3B causes individuals to be even 4 times more susceptible to periodontal disease.