METTL3-mediated RNA [m.sup.6]A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas

Introduction: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs.While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As [N.sup.6]-methyladenosine ([m.sup.6]A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. Methods: Protein expression of [m.sup.6]A regulators was measured by immunohistochemistry. Global levels and distribution of [m.sup.6]A methylation were separately analyzed by [m.sup.6]A enzyme-linked immunosorbent assay and [m.sup.6]A sequencing ([m.sup.6]A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its [m.sup.6]A- dependent regulatory mechanism in tumor progression and GH secretion. Results: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of [m.sup.6]A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45[gamma] act as the downstream targets in this process. Conclusion: Our findings strongly suggest that [m.sup.6]A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45[gamma] in a [m.sup.6]A-dependent manner.Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs. Key Words: pituitary adenoma, acromegaly, [N.sup.6]-methyladenosine, RNA methylation, METTL3