METTL3 通过介导 EZH2 的 M6A 修饰促进鼻咽癌的进展。
METTL3 promotes the progression of nasopharyngeal carcinoma through mediating M6A modification of EZH2.
机构信息
Department of Head and Neck Surgery, Linyi Cancer Hospital, Linyi, China.
出版信息
Eur Rev Med Pharmacol Sci. 2020 Apr;24(8):4328-4336. doi: 10.26355/eurrev_202004_21014.
OBJECTIVE
The aim of this study was to investigate whether METTL3 promoted the progression of nasopharyngeal carcinoma (NPC) by silencing CDKN1C through EZH2.
PATIENTS AND METHODS
Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression level of METTL3 in 48 pairs of NPC tissues and adjacent normal tissues. METTL3 expression in patients with different tumor lymph node metastasis (TNM) stages was detected by qRT-PCR as well. The Kaplan-Meier method was used to analyze the interplay between METTL3 expression and the prognosis of patients with NPC. At the same time, METTL3 expression in normal epithelial cell line (BEAS-2B) and NPC cell lines (SUNE-1 and C666-1) was examined using qRT-PCR. After METTL3 was knocked down in SUNE-1 cells, cell viability and migration abilities were analyzed by cell counting kit-8 (CCK-8) test and wound healing assay, respectively. The mRNA and protein expressions of EZH2 were detected by qRT-PCR and Western blot, respectively. RNA immunoprecipitation (RIP) assay was applied to detect the binding of METTL3 to EZH2 mRNA and the m6A modification on EZH2 mRNA. After knockdown of EZH2 in SUNE-1 cells, qRT-PCR was used to detect the mRNA expression of CDKN1C. Meanwhile, chromatin immunoprecipitation (ChIP) assay was conducted to analyze the binding of EZH2 to the CDKN1C promoter region. After down-regulation of METTL3 in SUNE-1 cells, the protein expressions of EZH2 and CDKN1C were detected using Western blot. After simultaneous knockdown of METTL3 and CDKN1C in SUNE-1 cells, CCK8 assay and wound healing assay were applied to examine cell viability and migration abilities.
RESULTS
METTL3 expression in NPC tissues was remarkably higher than that of adjacent normal tissues. Meanwhile, METTL3 expression in T3 and T4 tumors was significantly higher than that of T1 and T2 tumors. In patients with lymph node metastasis, the expression of METTL3 was remarkably higher than those without metastasis. Survival analysis demonstrated that patients with higher expression of METTL3 exhibited significantly longer overall survival time than those with lower METTL3 expression. QRT-PCR revealed that METTL3 was highly expressed in NPC cell lines, including SUNE-1 and C666-1. After knock-down of METTL3 in SUNE-1 cells, cell viability and migration abilities were both markedly weakened. Meanwhile, the protein expression of EZH2 was remarkably reduced. However, no significant changes were observed in EZH2 mRNA level. RIP assay revealed that METTL3 could bind to EZH2 mRNA, and a m6A modification was verified on EZH2 mRNA. After knockdown of EZH2, the mRNA level of CDKN1C in SUNE-1 cells was significantly up-regulated. CHIP assay indicated that EZH2 could bind to CDKN1C. Western blot showed that, after interfering with METTL3 in SUNE-1 cells, the protein expression of EZH2 decreased significantly, while CDKN1C was up-regulated. In addition, simultaneous downregulation of METTL3 and CDKN1C in SUNE-1 cells reversed the influence of METTL3 on cell viability and migration abilities.
CONCLUSIONS
METTL3 was highly expressed in NPC tissues, which might inhibit EZH2 expression by mediating M6A modification of EZH2 mRNA. Furthermore, CDKN1C could increase the malignancy of NPC cells and promote the progression of NPC.
目的
本研究旨在探讨 METTL3 是否通过 EZH2 介导的 CDKN1C 沉默来促进鼻咽癌（NPC）的进展。
方法
通过定量实时聚合酶链反应（qRT-PCR）检测 48 对 NPC 组织和相邻正常组织中 METTL3 的表达水平。同样通过 qRT-PCR 检测不同肿瘤淋巴结转移（TNM）阶段患者的 METTL3 表达。采用 Kaplan-Meier 法分析 METTL3 表达与 NPC 患者预后的相互作用。同时，通过 qRT-PCR 检测正常上皮细胞系（BEAS-2B）和 NPC 细胞系（SUNE-1 和 C666-1）中 METTL3 的表达。在 SUNE-1 细胞中敲低 METTL3 后，通过细胞计数试剂盒-8（CCK-8）试验和划痕愈合试验分别分析细胞活力和迁移能力。通过 qRT-PCR 和 Western blot 分别检测 EZH2 的 mRNA 和蛋白表达。应用 RNA 免疫沉淀（RIP）试验检测 METTL3 与 EZH2 mRNA 的结合以及 EZH2 mRNA 上的 m6A 修饰。在 SUNE-1 细胞中敲低 EZH2 后，通过 qRT-PCR 检测 CDKN1C 的 mRNA 表达。同时，进行染色质免疫沉淀（ChIP）试验分析 EZH2 与 CDKN1C 启动子区域的结合。在 SUNE-1 细胞中下调 METTL3 后，通过 Western blot 检测 EZH2 和 CDKN1C 的蛋白表达。在 SUNE-1 细胞中同时敲低 METTL3 和 CDKN1C 后，通过 CCK8 试验和划痕愈合试验检测细胞活力和迁移能力。
结果
NPC 组织中 METTL3 的表达明显高于相邻正常组织。同时，T3 和 T4 肿瘤中的 METTL3 表达明显高于 T1 和 T2 肿瘤。有淋巴结转移的患者中，METTL3 的表达明显高于无转移的患者。生存分析表明，METTL3 表达较高的患者总生存时间明显长于 METTL3 表达较低的患者。qRT-PCR 显示 METTL3 在 NPC 细胞系，包括 SUNE-1 和 C666-1 中高度表达。在 SUNE-1 细胞中敲低 METTL3 后，细胞活力和迁移能力均明显减弱。同时，EZH2 的蛋白表达明显降低。然而，EZH2 mRNA 水平没有明显变化。RIP 试验表明 METTL3 可以与 EZH2 mRNA 结合，并在 EZH2 mRNA 上验证了 m6A 修饰。在 SUNE-1 细胞中敲低 EZH2 后，CDKN1C 的 mRNA 水平明显上调。ChIP 试验表明 EZH2 可以与 CDKN1C 结合。Western blot 显示，在 SUNE-1 细胞中干扰 METTL3 后，EZH2 的蛋白表达明显下降，而 CDKN1C 则上调。此外，在 SUNE-1 细胞中同时敲低 METTL3 和 CDKN1C 可逆转 METTL3 对细胞活力和迁移能力的影响。
结论
METTL3 在 NPC 组织中高表达，可能通过介导 EZH2 mRNA 的 m6A 修饰抑制 EZH2 表达。此外，CDKN1C 可增加 NPC 细胞的恶性程度并促进 NPC 的进展。
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