Abstract：
OBJECTIVE: N6-methyladenosine (M6A) is the most prevalent epigenetic alteration. Methyltransferase-like 3 (METTL3) is a key player in the control of M6A modification. Methyltransferase promote the processing of mature miRNA in an M6A-dependent manner, thereby participating in disease occurrence and development. However, the regulatory mechanism of M6A in NK/T cell lymphoma (NKTCL) remains unclear.
METHODS: We determined the expression of METTL3 and its correlation with clinicopathological features using qRT-PCR and immunohistochemistry. We evaluated the effects of METTL3 on NKTCL cells using dot blot assay, CCK8 assay and subcutaneous xenograft experiment. We then applied M6A sequencing combined with gene expression omnibus data to screen candidate targets of METTL3. Finally, we investigated the regulatory mechanism of METTL3 in NKTCL by methylated RNA immunoprecipitation and RNA immunoprecipitation (RIP) assays.
RESULTS: We demonstrated that METTL3 was highly expressed in NKTCL cells and tissues and indicated poor prognosis. The METTL3 expression was associated with NKTCL survival. Functionally, METTL3 promoted the proliferation capability of NKTCL cells in vitro and in vivo. Furthermore, EBV-miR-BART3-3p was identified as the downstream effector of METTL3, and silencing EBV-miR-BART3-3p inhibited the proliferation of NKTCL. Finally, we confirmed that PLCG2 as a target gene of EBVmiR-BART3-3p by relative assays.
CONCLUSIONS: We identified that METTL3 is significantly up-regulated in NKTCL and promotes NKTCL development. M6A modification contributes to the progression of NKTCL via the METTL3/EBV-miR-BART3-3p/PLCG2 axis. Our study is the first to report that M6A methylation has a critical role in NKTCL oncogenesis, and could be a potential target for NKTCL treatment.
METHODS: We determined the expression of METTL3 and its correlation with clinicopathological features using qRT-PCR and immunohistochemistry. We evaluated the effects of METTL3 on NKTCL cells using dot blot assay, CCK8 assay and subcutaneous xenograft experiment. We then applied M6A sequencing combined with gene expression omnibus data to screen candidate targets of METTL3. Finally, we investigated the regulatory mechanism of METTL3 in NKTCL by methylated RNA immunoprecipitation and RNA immunoprecipitation (RIP) assays.
RESULTS: We demonstrated that METTL3 was highly expressed in NKTCL cells and tissues and indicated poor prognosis. The METTL3 expression was associated with NKTCL survival. Functionally, METTL3 promoted the proliferation capability of NKTCL cells in vitro and in vivo. Furthermore, EBV-miR-BART3-3p was identified as the downstream effector of METTL3, and silencing EBV-miR-BART3-3p inhibited the proliferation of NKTCL. Finally, we confirmed that PLCG2 as a target gene of EBVmiR-BART3-3p by relative assays.
CONCLUSIONS: We identified that METTL3 is significantly up-regulated in NKTCL and promotes NKTCL development. M6A modification contributes to the progression of NKTCL via the METTL3/EBV-miR-BART3-3p/PLCG2 axis. Our study is the first to report that M6A methylation has a critical role in NKTCL oncogenesis, and could be a potential target for NKTCL treatment.
摘要：
目的：N6-甲基腺苷（M6A）是最普遍的表观遗传改变。甲基转移酶样3(METTL3)是控制M6A修饰的关键角色。甲基转移酶以M6A依赖性方式促进成熟miRNA的加工，从而参与疾病的发生和发展。然而,M6A在NK/T细胞淋巴瘤（NKTCL）中的调控机制尚不清楚。
方法：我们使用qRT-PCR和免疫组织化学方法确定了METTL3的表达及其与临床病理特征的相关性。我们使用斑点印迹分析评估了METTL3对NKTCL细胞的影响，CCK8测定和皮下异种移植实验。然后，我们应用M6A测序结合基因表达综合数据来筛选METTL3的候选靶标。最后,我们通过甲基化RNA免疫沉淀和RNA免疫沉淀（RIP）分析研究了NKTCL中METTL3的调控机制。
结果：我们证明METTL3在NKTCL细胞和组织中高表达，提示预后不良。METTL3表达与NKTCL生存率相关。功能上,METTL3在体外和体内促进NKTCL细胞的增殖能力。此外，EBV-miR-BART3-3p被鉴定为METTL3的下游效应子，沉默EBV-miR-BART3-3p抑制NKTCL的增殖。最后,我们通过相关分析证实PLCG2是EBVmiR-BART3-3p的靶基因。
结论：我们发现METTL3在NKTCL中显著上调并促进NKTCL的发展。M6A修饰通过METTL3/EBV-miR-BART3-3p/PLCG2轴促进NKTCL的进展。我们的研究首次报道了M6A甲基化在NKTCL肿瘤发生中起关键作用，并可能成为NKTCL治疗的潜在靶点。
方法：我们使用qRT-PCR和免疫组织化学方法确定了METTL3的表达及其与临床病理特征的相关性。我们使用斑点印迹分析评估了METTL3对NKTCL细胞的影响，CCK8测定和皮下异种移植实验。然后，我们应用M6A测序结合基因表达综合数据来筛选METTL3的候选靶标。最后,我们通过甲基化RNA免疫沉淀和RNA免疫沉淀（RIP）分析研究了NKTCL中METTL3的调控机制。
结果：我们证明METTL3在NKTCL细胞和组织中高表达，提示预后不良。METTL3表达与NKTCL生存率相关。功能上,METTL3在体外和体内促进NKTCL细胞的增殖能力。此外，EBV-miR-BART3-3p被鉴定为METTL3的下游效应子，沉默EBV-miR-BART3-3p抑制NKTCL的增殖。最后,我们通过相关分析证实PLCG2是EBVmiR-BART3-3p的靶基因。
结论：我们发现METTL3在NKTCL中显著上调并促进NKTCL的发展。M6A修饰通过METTL3/EBV-miR-BART3-3p/PLCG2轴促进NKTCL的进展。我们的研究首次报道了M6A甲基化在NKTCL肿瘤发生中起关键作用，并可能成为NKTCL治疗的潜在靶点。
