Abstract A27: DNA methylation and transcriptional dysregulation of miRNA-137 and -193a in premalignant oral lesions and oral squamous cell carcinoma

Abstract

Abstract Background: Oral squamous cell carcinoma (OSCC) is a multistep process arising through the progressive accumulation of multiple genetic and epigenetic events. Tobacco, bidi (tobacco flakes wrapped in a tendu leaf) smoking and alcohol consumption are established risk factors associated with OSCC and premalignant oral lesions (PMOLs) in Indian population. The microRNAs (miRNAs) are non-coding RNAs that leads to gene silencing at the post- transcriptional level by degradation or repression of target mRNA. The miRNA expression may be oncogenic or tumor suppressor and can be regulated epigenetically, either through DNA methylation or histone modification. Tumor suppressor miRNA-137 and -193a are epigenetically silenced in many cancers. In this study, we attempted to elucidate the miRNA methylation and expression in the patient of PMOLs [leukoplakia with (LKP) or without dysplasia (LKPD) and oral submucous fibrosis (OSMF)] and OSCC to determine the potential role of both miRNA as early predictive biomarkers. Both miRNAs methylation was finally correlated with the clinicopathological variables in Indian population. Material and Methods: Methylation-specific PCR for miR-137 and -193a methylation was performed in biopsy proven tissues; controls (n=34), PMOLs (n=84) and OSCC (n=84) and in corresponding blood samples. Mature miRNA expression was examined using quantitative reverse transcriptase PCR in tissue samples (10 controls, 30 PMOLs and 10 OSCC) using TaqMan chemistry based primers and probes. RNU44 was selected for normalization as an endogenous control for expression analysis. All experiments were performed in triplicate in a clinical setting of a tertiary care hospital. Results: We observed miR-137 methylation frequency of 48% in tissue and 35% in blood samples of OSCC, and 27% in tissue and 10% in blood samples of PMOL group, as compared to the control samples which showed methylation frequency of 17.6% in tissue and 2.9% in blood samples. Further, we found methylation of miR-193a in patients with OSCC, i.e., 49% in tissue and 39% in blood samples of OSCC group. In PMOL group, methylation frequency of miR-193a was observed in 27% of tissue samples and 10% of blood samples, respectively as compared to the controls which showed miR-193a methylation frequency of 14.7% in tissue and 2.9% in blood samples. The promoter methylation frequency was found higher in histologically well-differentiated OSCC as compared to moderately and poorly differentiated OSCC. Multinomial logistic regression showed tobacco and bidi smoking were significantly associated with methylation of both miRNAs in PMOLs and OSCC group. As compared to controls, significant downregulation of miRNA-137 was observed only in OSCC group (2.23 fold, p=0.049) whereas miRNA-193a was significantly downregulated in both PMOL (LKP: 3.45 fold, p=0.000; LKPD: 3.80 fold, p=0.034) and OSCC group (4.34 fold, p=0.002). Conclusion: Our results suggest that promoter DNA methylation of both miRNA-137 and -193a in tissue and corresponding blood samples of PMOLs and OSCC group may be predictive biomarkers for early detection and malignant risk in premalignant lesions. Taken together DNA methylation and miRNA downregulation, we concluded that both miRNAs are epigenetically silenced during oral carcinogenesis. Note: This abstract was not presented at the conference. Citation Format: Vikram Bhatia, Madhu Mati Goel, Annu Makker, S P Agarwal, Sandeep Kumar, Sudhir K Goel. DNA methylation and transcriptional dysregulation of miRNA-137 and -193a in premalignant oral lesions and oral squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A27.