m6A 修饰的 BACE1-AS 通过 TUFT1 依赖性激活 Wnt 信号促进结直肠癌肝转移和干性样特性。

BACKGROUND: Liver metastasis is one of the most important reasons for high mortality of colorectal cancer (CRC). Growing evidence illustrates that lncRNAs play a critical role in CRC liver metastasis. Here we described a novel function and mechanisms of BACE1-AS promoting CRC liver metastasis. METHODS: qRT-PCR and in situ hybridization were performed to examine the BACE1-AS level in CRC. IGF2BP2 binding to m6A motifs in BACE1-AS was determined by RIP assay and S1m-tagged immunoprecipitation. Transwell assay and liver metastasis mice model experiments were performed to examine the metastasis capabilities of BACE1-AS knockout cells. Stemness-like properties was examined by tumor sphere assay and the expression of stemness biomarkers. Microarray data were acquired to analyze the signaling pathways involved in BACE1-AS promoting CRC metastasis. RESULTS: BACE1-AS is the most up-regulated in metastatic CRC associated with unfavorable prognosis. Sequence blast revealed two m6A motifs in BACE1-AS. IGF2BP2 binding to these two m6A motifs is required for BACE1-AS boost in metastatic CRC. m6A modified BACE1-AS drives CRC cells migration and invasion and liver metastasis both in vitro and in vivo. Moreover, BACE1-AS maintains the stemness-like properties of CRC cells. Mechanically, BACE1-AS promoted TUFT1 expression by ceRNA network through miR-214-3p. CRC patients with such ceRNA network suffer poorer prognosis than ceRNA-negative patients. Depletion of TUFT1 mimics BACE1-AS loss. BACE1-AS activated Wnt signaling pathway in a TUFT1 dependent manner. BACE1-AS/miR-214-3p/TUFT1/Wnt signaling regulatory axis is essential for CRC liver metastasis. Pharmacologic inhibition of Wnt signaling pathway repressed liver metastasis and stemness-like features in BACE1-AS over-expressed CRC cells. CONCLUSION: Our study demonstrated BACE1-AS as a novel target of IGF2BP2 through m6A modification. m6A modified BACE1-AS promotes CRC liver metastasis through TUFT1 dependent activation of Wnt signaling pathway. Thus, targeting BACE1-AS and its downstream Wnt signaling pathways may provide a new opportunity for metastatic CRC intervention and treatment.

背景：肝转移是结直肠癌（CRC）高死亡率的最重要原因之一。越来越多的证据表明，lncRNAs 在 CRC 肝转移中发挥着关键作用。在这里，我们描述了 BACE1-AS 促进 CRC 肝转移的新功能和机制。

方法：通过 qRT-PCR 和原位杂交检测 CRC 中 BACE1-AS 的水平。RIP 测定和 S1m 标记免疫沉淀测定 IGF2BP2 与 BACE1-AS 中 m6A 基序的结合。通过 Transwell 测定和肝转移小鼠模型实验检测 BACE1-AS 敲除细胞的转移能力。通过肿瘤球体测定和干细胞标志物的表达检测干性样特性。获取微阵列数据以分析涉及 BACE1-AS 促进 CRC 转移的信号通路。

结果：BACE1-AS 在转移性 CRC 中表达上调最明显，与不良预后相关。序列比对显示 BACE1-AS 中有两个 m6A 基序。IGF2BP2 与这两个 m6A 基序的结合对于 BACE1-AS 在转移性 CRC 中的促进作用是必需的。m6A 修饰的 BACE1-AS 驱动 CRC 细胞在体外和体内的迁移和侵袭以及肝转移。此外，BACE1-AS 维持 CRC 细胞的干性样特性。在机制上，BACE1-AS 通过 ceRNA 网络通过 miR-214-3p 促进 TUFT1 的表达。具有这种 ceRNA 网络的 CRC 患者比 ceRNA 阴性患者预后更差。TUFT1 的耗竭模拟了 BACE1-AS 的缺失。BACE1-AS 以 TUFT1 依赖的方式激活 Wnt 信号通路。BACE1-AS/miR-214-3p/TUFT1/Wnt 信号调节轴对于 CRC 肝转移至关重要。Wnt 信号通路的药理学抑制抑制了 BACE1-AS 过表达 CRC 细胞中的肝转移和干性样特征。

结论：我们的研究表明 BACE1-AS 是 IGF2BP2 通过 m6A 修饰的新型靶标。m6A 修饰的 BACE1-AS 通过 TUFT1 依赖性激活 Wnt 信号通路促进 CRC 肝转移。因此，靶向 BACE1-AS 及其下游 Wnt 信号通路可能为转移性 CRC 的干预和治疗提供新的机会。