Gemcitabine is the first-line chemotherapy drug for cholangiocarcinoma (CCA), but acquired resistance has been frequently observed in CCA patients. To search for potential long noncoding RNAs (lncRNAs) involved in gemcitabine resistance, two gemcitabine resistant CCA cell lines were established and dysregulated lncRNAs were identified by lncRNA microarray. Long intergenic non-protein coding RNA 665 (LINC00665) were found to rank the top 10 upregulated lncRNAs in our study, and high LINC00665 expression was closely associated with poor prognosis and chemoresistance of CCA patients. Silencing LINC00665 in gemcitabine resistant CCA cells impaired gemcitabine tolerance, while enforced LINC00665 expression increased gemcitabine resistance of sensitive CCA cells. The gemcitabine resistant CCA cells showed increased EMT and stemness properties, and silencing LINC00665 suppressed sphere formation, migration, invasion and expression of EMT and stemness markers. In addition, Wnt/β-Catenin signaling was activated in gemcitabine resistant CCA cells, but LINC00665 knockdown suppressed Wnt/β-Catenin activation. B-cell CLL/lymphoma 9-like (BCL9L), the nucleus transcriptional regulators of Wnt/β-Catenin signaling, plays a key role in the nucleus translocation of β-Catenin and promotes β-Catenin-dependent transcription. In our study, we found that LINC00665 regulated BCL9L expression by acting as a molecular sponge for miR-424-5p. Moreover, silencing BCL9L or miR-424-5p overexpression suppressed gemcitabine resistance, EMT, stemness and Wnt/β-Catenin activation in resistant CCA cells. In conclusion, our results disclosed the important role of LINC00665 in gemcitabine resistance of CCA cells, and provided a new biomarker or therapeutic target for CCA treament.

吉西他滨是胆管癌 (CCA) 的一线化疗药物，但在 CCA 患者中经常观察到获得性耐药。为了寻找与吉西他滨耐药相关的潜在长链非编码 RNA (lncRNA)，建立了两个对吉西他滨耐药的 CCA 细胞系，并通过 lncRNA 微阵列鉴定了失调的 lncRNA。在我们的研究中发现长基因间非蛋白质编码 RNA 665（LINC00665）在上调 lncRNA 中排名前 10，并且 LINC00665 的高表达与 CCA 患者的不良预后和化疗耐药性密切相关。在吉西他滨抗性 CCA 细胞中沉默 LINC00665 会损害吉西他滨的耐受性，而强制的 LINC00665 表达会增加敏感 CCA 细胞对吉西他滨的抗性。吉西他滨抗性 CCA 细胞显示出增加的 EMT 和干性特性，和沉默 LINC00665 抑制球体形成、迁移、侵袭和 EMT 和干性标记的表达。此外，Wnt/β-Catenin 信号在吉西他滨抗性 CCA 细胞中被激活，但 LINC00665 敲低抑制了 Wnt/β-Catenin 激活。B 细胞 CLL/淋巴瘤 9 样 (BCL9L) 是 Wnt/β-Catenin 信号传导的细胞核转录调节因子，在 β-Catenin 的细胞核易位中起关键作用并促进 β-Catenin 依赖性转录。在我们的研究中，我们发现 LINC00665 通过充当 miR-424-5p 的分子海绵来调节 BCL9L 表达。此外，沉默 BCL9L 或 miR-424-5p 过表达可抑制抗性 CCA 细胞中的吉西他滨抗性、EMT、干性和 Wnt/β-Catenin 激活。总之，我们的结果揭示了 LINC00665 在 CCA 细胞吉西他滨抗性中的重要作用，