The two-faced role of RNA methyltransferase METTL3 on cellular response to cisplatin in head and neck squamous cell carcinoma in vitro model.

Background: RNA methyltransferase-like 3 (METTL3) is responsible for methyl group transfer in the progression of N 6-methyladenosine (m6A) modification. This epigenetic feature contributes to the structural and functional regulation of RNA and consequently may promote tumorigenesis, tumor progression, and cellular response to anticancer treatment (chemo-, radio-, and immunotherapy). In head and neck squamous cell carcinoma (HNSCC), the commonly used chemotherapy is cisplatin. Unfortunately, cisplatin resistance is still a major cause of tumor relapse and patients' death. Thus, this study aimed to investigate the role of METTL3 on cellular response to cisplatin in HNSCC in vitro models. Materials and methods: HNSCC cell lines (H103, FaDu, and Detroit-562) with stable METTL3 knockdown (sgMETTL3) established with CRISPR-Cas9 system were treated with 0.5 tolerable plasma level (TPL) and 1 TPL of cisplatin. Further, cell cycle distribution, apoptosis, CD44/CD133 surface marker expression, and cell's ability to colony formation were analyzed in comparison to controls (cells transduced with control sgRNA). Results: The analyses of cell cycle distribution and apoptosis indicated a significantly higher percentage of cells with METTL3 knockdown 1) arrested in the G2/S phase and 2) characterized as a late apoptotic or death in comparison to control. The colony formation assay showed intensified inhibition of a single cell's ability to grow into a colony in FaDu and Detroit-562 METTL3-deficient cells, while a higher colony number was observed in H103 METTL3 knockdown cells after cisplatin treatment. Also, METTL3 deficiency significantly increased cancer stem cell markers' surface expression in all studied cell lines. Conclusion: Our findings highlight the significant influence of METTL3 on the cellular response to cisplatin, suggesting its potential as a promising therapeutic target for addressing cisplatin resistance in certain cases of HNSCC.

Understanding Hypoxia-Driven Tumorigenesis: The Interplay of HIF1A, DNA Methylation, and Prolyl Hydroxylases in Head and Neck Squamous Cell Carcinoma.

Hypoxia-inducible factor 1-alpha (HIF1A) is a key transcription factor aiding tumor cells' adaptation to hypoxia, regulated by the prolyl hydroxylase family (EGLN1-3) by directing toward degradation pathways. DNA methylation potentially influences EGLN and HIF1A levels, impacting cellular responses to hypoxia. We examined 96 HNSCC patients and three cell lines, analyzing gene expression of EGLN1-3, HIF1A, CA9, VEGF, and GLUT1 at the mRNA level and EGLN1 protein levels. Methylation levels of EGLNs and HIF1A were assessed through high-resolution melting analysis. Bioinformatics tools were employed to characterize associations between EGLN1-3 and HIF1A expression and methylation. We found significantly higher mRNA levels of EGLN3, HIF1A, GLUT1, VEGF, and CA9 (p = 0.021; p < 0.0001; p < 0.0001; p = 0.004, and p < 0.0001, respectively) genes in tumor tissues compared to normal ones and downregulation of the EGLN1 mRNA level in tumor tissues (p = 0.0013). In HNSCC patients with hypermethylation of HIF1A in normal tissue, we noted a reduction in HIF1A mRNA levels compared to tumor tissue (p = 0.04). In conclusion, the differential expression of EGLN and HIF1A genes in HNSCC tumors compared to normal tissues influences patients' overall survival, highlighting their role in tumor development. Moreover, DNA methylation could be responsible for HIF1A suppression in the normal tissues of HNSCC patients.

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients.

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.

Head and Neck Squamous Cell Carcinoma: Epigenetic Landscape.

Head and neck squamous carcinoma (HNSCC) constitutes the sixth most prevalent cancer worldwide. The molecular pathogenesis of HNSCC includes disorders in cell cycle, intercellular signaling, proliferation, squamous cell differentiation and apoptosis. In addition to the genetic mutations, changes in HNSCC are also characterized by the accumulation of epigenetic alterations such as DNA methylation, histone modifications, non-coding RNA activity and RNA methylation. In fact, some of them may promote cancer formation and progression by controlling the gene expression machinery, hence, they could be used as biomarkers in the clinical surveillance of HNSCC or as targets for therapeutic strategies. In this review, we focus on the current knowledge regarding epigenetic modifications observed in HNSCC and its predictive value for cancer development.

Expression of mitochondrial superoxide dismutase in polymorphonuclear leukocytes from patients with type 1 diabetes with and without microvascular complications.

INTRODUCTION: One of the causes of impaired antioxidant response in patients with type 1 diabetes might be decreased expression of mitochondrial manganese superoxide dismutase (MnSOD). OBJECTIVES: The aim of this study was to evaluate the expression of MnSOD on transcript and protein levels in polymorphonuclear leukocytes (PMNLs) from patients with type 1 diabetes and analyze its association with microvascular complications. PATIENTS AND METHODS: The MnSOD expression was assessed in PMNLs from 46 patients with type 1 diabetes and 12 age- and sex -matched healthy subjects. The study group was divided into 2 subgroups: with and without microvascular complications. The MnSOD expression on the transcript level was evaluated by real -time quantitative polymerase chain reaction, while that on the protein level by Western blot analysis. RESULTS: A significant increase in the MnSOD transcript level was observed in all patients with diabetes with and without microvascular complications (P = 0.01, P = 0.02, respectively). The MnSOD protein level was higher in patients without microvascular complications compared with those with complications and the control group (P = 0.05, P = 0.03, respectively). The MnSOD expression was positively correlated with fasting plasma glucose and total cholesterol levels both at the transcript level (r = 0.4, P <0.05 for both correlations) and at the protein level (r = 0.3 and r = 0.4, respectively, P <0.05). CONCLUSIONS: Although an increased MnSOD transcript level in patients with type 1 diabetes suggests enhanced antioxidant mobilization in all diabetic patients, decreased levels of the MnSOD protein in PMNLs from patients with microvascular complications compared with those without complications indicates that patients with microvascular complications may have impaired antioxidant response.