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

Yize Li,Cui Pang,Hongmei Zhang,Hongyan Peng,Yongmei Zhao,Peng Jiang,Wendong Bai,Wenchao Liu,Jingyi Ren,Jiarui Zhang,Xuelian Feng

doi:10.3389/fonc.2022.824258

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 in vitro 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 in vivo and in vitro experiments confirmed that METTL14 is a negative regulator of ovarian cancer cell proliferation via TROAP expression and that m6A RNA methylation regulates TROAP mRNA stability. In conclusion, METTL14 overexpression decreased ovarian cancer proliferation by inhibition of TROAP expression via an m6A RNA methylation-dependent mechanism.

Highlights

N6-methyladenosine (m6A) RNA methylation is the most prevalent RNA modification in eukaryotic cells and regulates many functions, including RNA splicing, translocation, stability, and protein translation [1]
We found a significant association between the copy number variations (CNVs) of the 23 m6A RNA methylation regulators, including methyltransferase-like 14 (METTL14), and their mRNA expression levels (Figure 1A and Supplemental Figure S1)
To verify this relationship between CNVs and mRNA levels, we evaluated the expression of 10 key m6A RNA methylation regulators in normal and cancerous ovarian tissues

Summary

Introduction

N6-methyladenosine (m6A) RNA methylation is the most prevalent RNA modification in eukaryotic cells and regulates many functions, including RNA splicing, translocation, stability, and protein translation [1]. This modification is catalyzed by RNA methyltransferases known as writers, such as methyltransferase-like 14 (METTL14), and is removed by demethylases known as erasers, such as fat mass and obesity-associated protein (FTO). M6A is recognized by m6A-binding proteins known as readers, such as IGF2BPs, which promote RNA stability and translation [2] These proteins determine the dynamic and reversible regulation of m6A RNA modification [3], which can become disrupted if the expression or function of these proteins are altered, resulting in the development of human diseases and cancers [4]. A better understanding of these m6A writers, erasers, and readers will provide the information necessary to clarify the biological functions and potential mechanisms of m6A

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Results

Conclusion