Downregulation of Methyltransferase-Like 14 Promotes Ovarian Cancer Cell Proliferation Through Stabilizing TROAP mRNA

Abstract

Altered expression levels of the proteins that regulate N6-methyladenosine (m⁶A) RNA methylation, including methyltransferase-like 14 (METTL14), are associated with cancer development. Based on our analysis of m⁶A methylation regulators using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, we focused on the regulatory role of METTL14 in ovarian cancer. We performed bioinformatics and survival analyses with these datasets and also used METTL14-overexpressing SKOV-3 ovarian cancer cells for <i>in vitro</i> studies. Trophinin associated protein (TROAP) siRNA and treatment with or without actinomycin D was used in the cells for qRT-PCR, western blot, cDNA microarray, cell viability, colony formation, luciferase gene reporter, methylated RNA immunoprecipitation (MeRIP)-qPCR, total RNA methylation, and RNA stability assays. Additionally, ovarian cancer and normal tissue samples were analyzed by immunohistochemistry, qRT-PCR, and western blot assays. The TCGA and GEO data confirmed copy number variations (CNVs) of these m⁶A RNA methylation regulators in ovarian cancer tissues. Furthermore, reduced METTL14 expression was associated with alterations in CNVs as well as poor patient survival in ovarian cancer. Moreover, the METTL14 and m⁶A RNA methylation levels were both significantly reduced in ovarian cancer tissues than in normal tissues. Restoration of METTL14 expression suppresses ovarian cancer cell proliferation by inhibition of TROAP expression. Further <i>in vivo</i> and <i>in vitro</i> experiments confirmed that METTL14 is a negative regulator of ovarian cancer cell proliferation <i>via</i> TROAP expression and that m⁶A RNA methylation regulates TROAP mRNA stability. In conclusion, METTL14 overexpression decreased ovarian cancer proliferation by inhibition of TROAP expression <i>via</i> an m⁶A RNA methylation-dependent mechanism.