METTL14 介导的 ITGB4 mRNA 的 N6-甲基腺苷修饰抑制透明细胞肾细胞癌的转移。
METTL14-mediated N-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma.
机构信息
Department of Urology, First Hospital of China Medical University, No. 155 Nanjing North Street, Heping District, Shenyang City, 110004, Liaoning Province, People's Republic of China.
Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, 110004, Liaoning, People's Republic of China.
出版信息
Cell Commun Signal. 2022 Mar 19;20(1):36. doi: 10.1186/s12964-022-00831-5.
BACKGROUND
Integrin β4 (ITGB4) participates in tumorigenesis and progression of several malignancies, but its role and related mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear.
METHODS
Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were used to detect mRNA and protein levels of relevant genes. Biological functions of ITGB4 and methyltransferase-like 14 (METTL14) were determined by in vitro and in vivo experiments. The levels of N6-methyladenosine (m6A) in ccRCC tissues and adjacent normal tissues were calculated via total RNA m6A quantification assay. The m6A modification of ITGB4 was demonstrated via m6A RNA immunoprecipitation (MeRIP), RIP and luciferase reporter assays.
RESULTS
ITGB4 was significantly overexpressed in ccRCC tissues and high level of ITGB4 predicted poor prognosis as well as metastasis. Functionally, ITGB4 stimulated ccRCC cell migration and invasion in vitro and metastasis in vivo with epithelial-mesenchymal transition (EMT) strengthened. Mechanically, the total levels of m6A were reduced in ccRCC tissues. METTL14, a favorable factor for ccRCC patients' prognosis, facilitated m6A modification on ITGB4 3'UTR and subsequently accelerated ITGB4 mRNA degradation, leading to its declined expression. Furthermore, the METTL14-mediated inhibition of ITGB4 expression was dependent on the YTH domain family protein 2 (YTHDF2), which acted as an m6A reader to bind to ITGB4 mRNA and to promote its decay. In addition, we demonstrated that knockdown of METTL14 promoted ccRCC cell migration, invasiveness and metastasis as well as stimulating the EMT process and the PI3K/AKT signal by overexpressing ITGB4.
CONCLUSION
Our study reveals that METTL14 inhibits ITGB4 expression via m6A modification to attenuate metastasis and EMT of ccRCC cells, suggesting the METTL14/ITGB4 axis as a potential prognostic biomarker and therapeutic target for ccRCC. Video Abstract.
背景
整合素β4（ITGB4）参与了几种恶性肿瘤的发生和进展，但它在透明细胞肾细胞癌（ccRCC）中的作用和相关机制尚不清楚。
方法
采用实时定量 PCR（qRT-PCR）、Western blot 和免疫组织化学检测相关基因的 mRNA 和蛋白水平。通过体外和体内实验确定 ITGB4 和甲基转移酶样 14（METTL14）的生物学功能。通过总 RNA m6A 定量测定法计算 ccRCC 组织和相邻正常组织中的 N6-甲基腺苷（m6A）水平。通过 m6A RNA 免疫沉淀（MeRIP）、RIP 和荧光素酶报告基因实验证实了 ITGB4 的 m6A 修饰。
结果
ITGB4 在 ccRCC 组织中显著过表达，高水平的 ITGB4 预示着预后不良和转移。功能上，ITGB4 增强上皮-间充质转化（EMT），刺激 ccRCC 细胞在体外迁移和侵袭，并在体内转移。机制上，ccRCC 组织中的总 m6A 水平降低。METTL14 是 ccRCC 患者预后的有利因素，促进 ITGB4 3'UTR 的 m6A 修饰，进而加速 ITGB4 mRNA 的降解，导致其表达下降。此外，METTL14 介导的 ITGB4 表达抑制依赖于 YTH 结构域家族蛋白 2（YTHDF2），YTHDF2 作为 m6A 阅读器与 ITGB4 mRNA 结合并促进其降解。此外，我们证明通过过表达 ITGB4 ，METTL14 的敲低促进了 ccRCC 细胞的迁移、侵袭和转移，并刺激了 EMT 过程和 PI3K/AKT 信号。
结论
我们的研究表明，METTL14 通过 m6A 修饰抑制 ITGB4 的表达，从而减弱 ccRCC 细胞的转移和 EMT，提示 METTL14/ITGB4 轴作为 ccRCC 的潜在预后生物标志物和治疗靶点。
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