Expression And Molecular Mechanism Of 5hmC In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is the most common malignancy in oral and maxillofacial regions.Local recurrence and cervical lymph node metastasis are critical factors contributing to OSCC-related deaths.However,the mechanism of OSCC pathogenesis and invasion is still unclear.Therefore,it is difficult for early diagnosis and individualized treatment strategies.Methylation and methylation of DNA molecules have become the focus of epigenetic research in recent years.5-hm C(5-hydroxymethylcytosine)is considered to be an intermediate of active DNA demethylation.In addition to DNA demethylation,it also regulates genes related to development,pluripotency and rna splicing,and is related to malignant transformation of tumor.TET(Ten-Eleven Translocation)protein catalyzes the hydroxylation of 5m C to 5hm C utilized Fe2+ and alpha ketoparic acid,which is the main limiting factor for the activity of DNA demethylation.Recent studies have shown that different tet family members are reported as tumor suppressor in different tumor types.5hmc has been found to be lossing in many solid tumors which considered to be a biomarker for tumor recognition and progression.However,there are few reports in oral squamous cell carcinoma(OSCC).Objective:To characterize the expression patterns and oncogenic roles of 5hm C in oral squamous cell carcinoma and to determine the inhibitory effect of epigenetic drugs on oral squamous cell carcinoma.Methods:1.To characterize the abundance of 5hm C in OSCC tissue specimens by immunoflouorescence.To analyze the correlation between 5hmc expression level and clinical parameters.2.To detece the expression of 5hm C and TET2 in samples of 4NQO-induced animal model.3.The cell phenotype was verified by Western blot,Cck-8 assay,Flat clones assay and Transwell assays which were used to compare the role of OSCC cells in inhibit tumor cells proliferation migration,apoptosis,senescence,colony forma after transfection of overexpressed TET2 virus.4.The expression of 5hm C and TET2 were oberved by DNA methylation inhibitors 5-aza treatment.TETs expression of m RNA was also detected by reverse transcription polymerase chain reaction.Results: 1.We evaluated the expression patterns of 5hm C by immunohistochemical staining in a retrospective cohort of 95 primary OSCC samples.The results indicated 5hm C downregulated in these OSCC specimens,a clear indicative of aberrant lowexpression of 5hm C in a major fraction of OSCC samples(P<0.01).There were no significant correlations between 5hm C expression with patients’ age,gender,tumor size,local invasion,and clinical stage.Noticeably,significant association between 5hm C expression was associated with cervical nodes metastasis(P=0.0041)and pathological grade(P=0.0239).The Kaplan-Meier survival analyses indicated that high 5hm C level had adverse prognostic impact for patients’ survival as demonstrated that patients with high 5hm C significantly associated with reduced overall,disease-free and disease-specific survival as compared to those with low 5hm C(Log-rank,P = 0.0210,0.0313,0.0415,respectively).2.The expression of 5hm C and TET2 declined with disease progression in 4NQO-induced tongue Carcinmoma.3.5hm C expression was potently promoted after transfection of overexpressed TET2 virus.and inhibited cell proliferation,migration,invasion and tumorsphere formation.4.5aza robustly inhibited 5hm C and TET2 expression in OSCC cells in a dose—and time—dependent manner,as well as enhanced of TETs expression of m RNA by reverse transcription polymerase chain reaction.Conclusions: 5hm C is a new epigenetic marker in the progression of oral squamous cell carcinoma.It can inhibit tumor cell proliferation,migration and invasion,and participate in tumor development.TET2 gene is a key factor of 5hm C,which regulates the expression level of 5hm C.The DNA methyltransferase inhibitor 5-azacytidine(5-aza)can promote the expression of 5hm C to reverse the abnormal state of low expression,thus inhibiting the progress of oral squamous cell carcinoma,suggesting a new target for the treatment of oral squamous cell carcinoma.