DataSheet_1_Downregulation of Methyltransferase-Like 14 Promotes Ovarian Cancer Cell Proliferation Through Stabilizing TROAP mRNA.docx

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.

调控N6-甲基腺嘌呤（N6-methyladenosine, m6A）RNA甲基化的蛋白质（包括甲基转移酶样14（methyltransferase-like 14, METTL14））的表达水平异常与癌症的发生发展密切相关。本研究基于对癌症基因组图谱（The Cancer Genome Atlas, TCGA）与基因表达综合数据库（Gene Expression Omnibus, GEO）数据集内m6A RNA甲基化调控因子的分析，聚焦于METTL14在卵巢癌中的调控功能。我们针对上述数据集开展生物信息学与生存分析，并利用过表达METTL14的SKOV-3卵巢癌细胞系开展体外实验；实验中，我们采用靶向trophinin相关蛋白（Trophinin associated protein, TROAP）的小干扰RNA（small interfering RNA, siRNA），并设置添加与不添加放线菌素D（actinomycin D）的细胞处理组，随后开展实时定量反转录PCR（quantitative real-time reverse transcription PCR, qRT-PCR）、蛋白质免疫印迹（western blot）、cDNA微阵列、细胞活力检测、集落形成实验、荧光素酶报告基因实验、甲基化RNA免疫沉淀（methylated RNA immunoprecipitation, MeRIP）-qPCR、总RNA甲基化水平检测以及RNA稳定性实验。此外，我们通过免疫组织化学、qRT-PCR以及蛋白质免疫印迹实验，对卵巢癌组织与正常组织样本进行了分析。TCGA与GEO数据集证实，卵巢癌组织中存在上述m6A RNA甲基化调控因子的拷贝数变异（copy number variations, CNVs）。进一步分析显示，METTL14表达下调与拷贝数变异改变以及卵巢癌患者的不良预后显著相关。此外，卵巢癌组织中METTL14的表达水平与m6A RNA甲基化水平均显著低于正常对照组织。恢复METTL14的表达可通过抑制TROAP的表达，进而抑制卵巢癌细胞的增殖能力。后续体内与体外实验进一步证实，METTL14可通过调控TROAP的表达负向调控卵巢癌细胞增殖，且m6A RNA甲基化可直接调控TROAP的mRNA稳定性。综上，METTL14过表达可通过依赖m6A RNA甲基化的机制抑制TROAP的表达，从而降低卵巢癌细胞的增殖能力。