Department of Thoracic Surgery, Shanghai Tongji Hospital, School of Medicine, Tongji University, Shanghai, P.R. China.
Clin Transl Med. 2021 Sep;11(9):e545. doi: 10.1002/ctm2.545.
Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and treatment-resistant tumor. The biological implications and molecular mechanism of cancer stem-like cells (CSCs) in ESCC, which contribute to therapeutic resistance such as radioresistance, remain elusive.
Quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and in situ hybridization assays were used to detect methyltransferase-like 14 miR-99a-5p tribble 2 (METTL14/miR-99a-5p/TRIB2) expression in ESCC. The biological functions of METTL14/miR-99a-5p/TRIB2 were demonstrated in vitro and in vivo. Mass spectrum analysis was used to identify the downstream proteins regulated by TRIB2. Chromatin immunoprecipitation (IP), IP, N -methyladenosine (m A)-RNA IP, luciferase reporter, and ubiquitination assays were employed to explore the molecular mechanisms underlying this feedback circuit and its downstream pathways.
We found that miR-99a-5p was significantly decreased in ESCC. miR-99a-5p inhibited CSCs persistence and the radioresistance of ESCC cells, and miR-99a-5p downregulation predicted an unfavorable prognosis of ESCC patients. Mechanically, we unveiled a METTL14-miR-99a-5p-TRIB2 positive feedback loop that enhances CSC properties and radioresistance of ESCC cells. METTL14, an m A RNA methyltransferase downregulated in ESCC, suppresses TRIB2 expression via miR-99a-5p-mediated degradation of TRIB2 mRNA by targeting its 3' untranslated region, whereas TRIB2 induces ubiquitin-mediated proteasomal degradation of METTL14 in a COP1-dependent manner. METTL14 upregulates miR-99a-5p by modulating m A-mediated, DiGeorge critical region 8-dependent pri-mir-99a processing. Hyperactivation of TRIB2 resulting from this positive circuit was closely correlated with radioresistance and CSC characteristics. Furthermore, TRIB2 activates HDAC2 and subsequently induces p21 epigenetic repression through Akt/mTOR/S6K1 signaling pathway activation. Pharmacologic inhibition of HDAC2 effectively attenuates the TRIB2-mediated effect both in vitro and in patient-derived xenograft models.
Our data highlight the presence of the METTL14/miR-99a-5p/TRIB2 axis and show that it is positively associated with CSC characteristics and radioresistance of ESCC, suggesting potential therapeutic targets for ESCC treatment.
食管鳞状细胞癌（ESCC）是一种侵袭性强、治疗耐药的肿瘤。癌症干细胞样细胞（CSCs）在 ESCC 中对治疗耐药（如放射耐药）的生物学意义和分子机制仍不清楚。
采用定量实时聚合酶链反应、western blot、免疫组织化学和原位杂交检测 ESCC 中甲基转移酶样 14 miR-99a-5p 三叶草 2（METTL14/miR-99a-5p/TRI B2）的表达。在体外和体内证明了 METTL14/miR-99a-5p/TRI B2 的生物学功能。质谱分析用于鉴定 TRI B2 调节的下游蛋白。染色质免疫沉淀（Ch IP）、Ch IP、N-甲基腺苷（m A）-RNA Ch IP、荧光素酶报告基因和泛素化测定用于探索该反馈回路及其下游途径的分子机制。
我们发现 miR-99a-5p 在 ESCC 中显著下调。miR-99a-5p 抑制 ESCC 细胞的 CSCs 持久性和放射耐药性，miR-99a-5p 下调预示 ESCC 患者预后不良。在机制上，我们揭示了一个 METTL14-miR-99a-5p-TRIB2 正反馈环，该环增强了 ESCC 细胞的 CSC 特性和放射耐药性。METTL14 是一种在 ESCC 中下调的 m A RNA 甲基转移酶，通过 miR-99a-5p 介导的 TRIB2 mRNA 降解来抑制 TRIB2 表达，该过程靶向其 3'非翻译区，而 TRIB2 以 COP1 依赖的方式诱导 METTL14 的泛素介导的蛋白酶体降解。METTL14 通过调节 m A 介导的、DiGeorge 关键区域 8 依赖性 pri-miR-99a 加工来上调 miR-99a-5p。该正回路的过度激活与放射耐药性和 CSC 特征密切相关。此外，TRIB2 通过激活 Akt/mTOR/S6K1 信号通路激活来激活 HDAC2，随后诱导 p21 的表观遗传抑制。通过药理学抑制 HDAC2，可有效减轻体外和患者来源的异种移植模型中 TRIB2 介导的作用。
我们的数据突出了 METTL14/miR-99a-5p/TRI B2 轴的存在，并表明其与 ESCC 的 CSC 特征和放射耐药性呈正相关，提示 ESCC 治疗的潜在治疗靶点。
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