Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 250021, Jinan, China.
The Hepatosplenic Surgery Center, the First Affiliated Hospital of Harbin Medical University, 150001, Harbin, China.
Cell Death Dis. 2020 Aug 15;11(8):637. doi: 10.1038/s41419-020-02896-x.
Neuropilin-1 regulated by miR-320a participates in the progression of cholangiocarcinoma by serving as a co-receptor that activates multiple signaling pathways. The present study sought to investigate upstream lncRNAs that control the expression of miR-320a/neuropilin-1 axis and dissect some of the underlying mechanisms. Here we report lncRNA TTN-AS1 (titin-antisense RNA1) acts as a sponging ceRNA to downregulate miR-320a and is highly expressed in human cholangiocarcinoma tissues and cells. The expression of the above three molecules is correlated with the clinicopathologic parameters of cholangiocarcinoma patients. In this study, multiple bioinformatics tools and databases were employed to seek potential lncRNAs that have binding sites with miR-320a and TTN-AS1 was identified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile duct epithelial cells. The regulatory role of TTN-AS1 on miR-320a was further evaluated by luciferase reporter and RNA pulldown assays, coupled with in situ hybridization and RNA immunoprecipitation analyses, which showed that TTN-AS1 bound to miR-320a through an argonaute2-dependent RNA interference pathway in the cytoplasm of cholangiocarcinoma cells. Knockdown and overexpression assays showed that the regulatory effect between TTN-AS1 and miR-320 was in a one-way manner. TTN-AS1 promoted the proliferation and migration of cholangiocarcinoma cells via the miR-320a/ neuropilin-1 axis. The function of TTN-AS1 on tumor growth and its interaction with miR-320a were confirmed in animal models. Further mechanistic studies revealed that TTA-AS1, through downregulating miR-320a, promoted cell cycle progression, epithelial-mesenchymal transition, and tumor angiogenesis by upregulating neuropilin-1, which co-interacted with the hepatocyte growth factor/c-Met and transforming growth factor (TGF)-β/TGF-β receptor I pathways. In conclusion, the present results demonstrate that lncRNA TTA-AS1 is a sponging ceRNA for miR-320a, which in turn downregulates neuropilin-1 in cholangiocarcinoma cells, indicating these three molecules represent potential biomarkers and therapeutic targets in the management of cholangiocarcinoma.
神经纤毛蛋白 1 通过充当激活多条信号通路的共受体，受 miR-320a 调控，参与胆管癌的进展。本研究旨在探讨调控 miR-320a/神经纤毛蛋白 1 轴表达的上游长链非编码 RNA，并剖析部分潜在机制。本研究报告称，反义肌联蛋白 TTN-AS1（titin-antisense RNA1）作为 ceRNA 下调 miR-320a，在人胆管癌组织和细胞中高表达。上述三种分子的表达与胆管癌患者的临床病理参数相关。在这项研究中，我们使用了多种生物信息学工具和数据库来寻找与 miR-320a 具有结合位点的潜在长链非编码 RNA，TTN-AS1 被鉴定出来，因为它在胆管癌和正常胆管上皮细胞之间的变化幅度最大。通过荧光素酶报告和 RNA 下拉测定、原位杂交和 RNA 免疫沉淀分析进一步评估了 TTN-AS1 对 miR-320a 的调控作用，结果表明，在胆管癌细胞的细胞质中，TTN-AS1 通过依赖 Argonaute2 的 RNA 干扰途径与 miR-320a 结合。敲低和过表达实验表明，TTN-AS1 与 miR-320 之间的调控作用是单向的。TTN-AS1 通过 miR-320a/神经纤毛蛋白 1 轴促进胆管癌细胞的增殖和迁移。在动物模型中证实了 TTN-AS1 对肿瘤生长的功能及其与 miR-320a 的相互作用。进一步的机制研究表明，TTN-AS1 通过下调 miR-320a，通过上调共与肝细胞生长因子/c-Met 和转化生长因子（TGF）-β/TGF-β 受体 I 通路相互作用的神经纤毛蛋白 1，促进细胞周期进程、上皮-间充质转化和肿瘤血管生成。总之，本研究结果表明，长链非编码 RNA TTN-AS1 是 miR-320a 的海绵 ceRNA，进而下调胆管癌细胞中的神经纤毛蛋白 1，表明这三种分子是胆管癌管理中的潜在生物标志物和治疗靶点。
