Aberrant N6 Messenger RNA Methylation (m 6methyladenosine) in oral cancer

Abstract

Background and Objectives : RNA modification by methylation at the 6h nitrogen atom of the adenosine base (or m6A) within the internal mRNA sequence is regulated by two main enzymes, methyltransferase-like 3 (METTL3) and 14 (METTL14). Defects in the m6A modification are associated with initiation and progression of various cancers. However, the role of the m6A and its relevant enzymes, METTL3 and METTL14, in the molecular pathogenesis of oral squamous cell carcinoma (OSCC) has not yet been known. This study, therefore, aimed to determine expressions of METTL3 and METTL14 in OSCC specimens and to investigate in vitro aggressiveness of their aberrant expressions in OSCC cell lines.

Methods : METTL3 and METTL 14 expressions in 50 OSCC specimens and 11 normal oral tissues were examined by immunohistochemistry, and correlations of these protein expressions with clinicopathological characteristics were determined. mRNA and protein expressions of METTL3 and METTL14 were investigated in three OSCC cell lines, including HN5, HN6, and HN15, compared to those in normal primary human oral keratinocytes (HOKs) by RT-qPCR and Western blot hybridization, respectively. Moreover, m6A amounts were examined in these cell lines by colorimetry. Cell proliferation, migration, and invasion were studied by BrdU, wound healing, and Transwell chamber assays, respectively, after silencing of METTL3, METTL14 or both by siRNA transfection. RT-qPCR was also performed to detect the degrees of METTL3 and METTL 14 expressions after silencing. Results : Immunostaining of METTL3 and METTL14 was found to be localized in cancer cell nuclei. The mean percentages of METTL3- and METTL14-positive cells were significantly increased in OSCC tissues, compared to those in normal tissues (p<0.001). Based on histopathological diagnosis for OSCC severity, the percentages of METTL3- positive cells were strongly correlated with the decreased differentiation of OSCC cells (1-0.564, p<0.001), while a moderate correlation was found for the percentages of METTL 14-positive cells (r=0.316, p=0.013). Moreover, the percentages of METTL3- and METTL14-positive cells were correlated with the advanced pTNM stages and short five-year overall survival (p<0.05). Both mRNA and protein expressions of METTL3 and METTL14 were significantly increased only in HN6, compared to those in HOKs (p<0.05). The m'A amounts were also significantly increased in HN6 (p<0.05). Therefore, further transient transfection studies with an emphasis on METTL3 and METTL14 silencing were conducted in HN6. As expected, a significant decrease in METTL3 or METTL14 mRNA expression in HN6 was found after transfection with siRNA against each corresponding gene (p<0.001). Transfection with METTL3 siRNA or with both METTL3 and METTL14 siRNA significantly inhibited HN6 proliferation (p<O.01), but it failed to mitigate HN6 migration or invasion. Conclusions : Expressions of METTL3 and METTL14 were significantly enhanced in OSCC tissues and positively correlated with histopathological diagnosis for OSCC severity. Both METTL3 and METTL4 overexpression were associated with both advanced pTNM stages and short five-year overall survival in patients with OSCC. Significant overexpression of METTL3 and METTL 14 was also found in the OSCC cell line, HN6, which promoted cell proliferation, but not cell migration or invasion.