OSCC is the sixth most common malignancy worldwide, and its occurrence and development result from the gradual accumulation of various factors, including epigenetic factors. DNA methylation modifications affect genetic mutations and occur before tumor formation [31]. In general, the epigenetic state of cells is precisely regulated to maintain an appropriate state of differentiation. However, this carefully balanced genomic programming is disturbed in cancer, resulting in uncontrolled cell proliferation, impaired differentiation, and resistance to apoptosis [32].Therefore, the DNA methylation status of target genes is a potential therapeutic target in cancer treatment and biomarker in early detection and prognostication of cancer. Consequently, it should be explored for improving cancer treatment outcomes.
In this study, the methylation degree revealed that as the cancer became less differentiated, DNMT3A promoter methylation decreased gradually, indicating that its expression was no longer inhibited. The level of global methylation of cancer increased, thereby promoting the malignant transformation of tumors. DNMT3A is involved in the oncogenic process of various tumors by regulating the level of promoter methylation. Yu et al. revealed that miR-26a-5p targeted DNMT3A to reduce the degree of global methylation in non-small cell lung cancer and restore the SFRP1 expression, thereby regulating cell viability and the stem-like phenotype by regulating the Wnt/β-catenin pathway [33]. Lu et al. found DNMT3A promoted the Warburg effect and tumor malignant biological behavior by inhibiting the miR-603 expression. Therefore, it is a potential therapeutic target for ovarian cancer [34]. Pang et al. demonstrated that MYC-recruited DNMT3A induced promoter methylation, resulting in miR-200b silencing, thereby promoting epithelial mesenchymal transformation and mammary globular formation in triple-negative breast cancer cells [35].
In the present study, TET2 functioned as a tumor suppressor gene (TSG) in OSCC. The methylation degree revealed that TET2 was predominantly unmethylated in tissues with a lower degree of malignancy, indicating that the TET2 expression was not inhibited. Further, as the degree of differentiation decreased, the degree of promoter methylation increased, and the expression was inhibited. Promoter methylation of TSGs can be an early occurrence in the development of tumors. This epigenetic alteration may confer advantages to cell growth, in turn promoting malignant transformation. TSGs that undergo abnormal promoter hypermethylation can encode various protein products, including DNA repair factors, cell cycle inhibitors, and cell adhesion receptors [36]. Hitchins et al. reported that promoter hypermethylation of MLH1 and epigenetic silencing were associated with various cancers characterized by defects in mismatch repair [37]. Casalino et al. showed that CDKN2A encoded the cell cycle inhibitor p16INK4A, and loss of the p16INK4A expression because of hypermethylation of its promoter was an early event in breast and lung cancers [38]. Markou et al. selected five TSGs, involved in cancer cell differentiation, proliferation, apoptosis, adhesion, and metastasis for methylation evaluation, which in combination with circulating tumor cells and matched plasma-free DNA provided effective prognostic information for patients with early lung cancer [39]. These examples confirm that abnormal methylation contributes to the development of cancer by affecting genes involved in key cellular processes and suggest that the methylation degree of the promoter of TSGs change continuously during tumor development, which is an effective mean for the early detection, disease surveillance, and evaluation of the therapeutic efficacy.
The present study showed a higher protein expression of DNMT3A in tumor samples than in surrounding healthy tissue samples. The TET2 expression in cancer tissues was significantly reduced, and its recovery could effectively inhibit the malignant biological behavior of OSCC. Altered expression levels of DNMTs, particularly elevated DNMT3A levels, are common in multiple cancer samples and cell lines [40]. TET proteins regulate the equilibrium between DNA methylation and demethylation by regulating the dynamic transformation among cytosine, 5-mC, and 5-hmC [41]. However, missense and truncated TET mutations have been reported in nearly all types of solid tumors [42], and their reduced protein expression and 5-hmC levels are markers of many cancer types, including colorectal cancer [43], glioblastoma [44], cervical cancer [45], and pancreatic cancer [46]. The aforementioned studies are consistent with our findings, showing that modulating the DNMT3A and TET2 expression levels could effectively change the biological behavior of OSCC.
This was the first study to show that DNMT3A and TET2 methylation levels change with the degree of cancer differentiation and that DNMT3A knockdown or TET2 overexpression could effectively inhibit the proliferation and migration of OSCC. Although the oncogenic mechanisms of DNMT3A and TET2 should be further explored, they have a wide range of applications as potential therapeutic targets for cancer.