Breast cancer (BC) has a marked tendency to spread to the bone, resulting in significant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown. Bone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofluorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isolation by RNA purification and enzyme-linked immunosorbent assays. We identified that a novel circRNA, circIKBKB, was upregulated significantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dramatically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKβ-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promoters of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB flanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis effectively. We revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis.

中文翻译：

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环状RNA circIKBKB通过维持NF-κB/骨重塑因子信号促进乳腺癌骨转移

乳腺癌 (BC) 具有明显的向骨骼扩散的趋势，导致严重的骨骼并发症和死亡率。最近，据报道环状 RNA (circRNA) 有助于癌症的发生和进展。然而，circRNAs 在 BC 骨转移（BC-BM）中的功能和机制仍然很大程度上未知。使用 circRNA 深度测序筛选骨转移性 circRNA，并在有或没有骨转移的 BC 组织中使用原位杂交进行验证。circIKBKB 在诱导骨转移前生态位形成和骨转移中的作用是使用破骨细胞生成、免疫荧光和骨吸收坑试验确定的。使用 RNA 下拉研究了 circIKBKB 介导的 NF-κB/骨重塑因子信号传导和 EIF4A3 诱导的 circIKBKB 激活的机制，荧光素酶报告基因，通过 RNA 纯化和酶联免疫吸附测定分离染色质。我们发现一种新的 circRNA，circIKBKB，在骨转移 BC 组织中显着上调。通过在体内和体外促进破骨细胞生成，过表达 circIKBKB 显着增强了 BC 细胞诱导骨转移前生态位形成的能力。机械上，circIKBKB 通过促进 IKKβ 介导的 IκBα 磷酸化、抑制 IκBα 反馈环和促进 NF-κB 对多种骨重塑因子的启动子来激活 NF-κB 通路。此外，作为前 mRNA 剪接因子的 EIF4A3 通过直接与 circIKBKB 侧翼区域结合来促进 circIKBKB 的环化。重要的，抑制剂 eIF4A3-IN-2 治疗可降低 circIKBKB 表达并有效抑制乳腺癌骨转移。我们揭示了在骨转移性 BC 中 circIKBKB 介导的 NF-κB 过度活化的合理机制，这可能代表了治疗乳腺癌骨转移的潜在策略。