LncRNA LINC01315 silencing modulates cancer stem cell properties and epithelial-to-mesenchymal transition in colorectal cancer via miR-484/DLK1 axis

Long non-coding RNA long intergenic non-protein coding RNA 01315 (LncRNA LINC01315) has been found to be implicated in various cancers, but its role and functions in colorectal cancer (CRC) remain to be addressed. Data on LINC01315 expression in CRC were gathered using bioinformatics analysis, and cancer stem cells (CSCs) were sorted by aldehyde dehydrogenase (ALDH) assay and flow cytometry. Migration, invasion, and stemness of CSCs isolated from CRC cells after transfection were determined by scratch, Transwell, and sphere-formation assays, respectively. Tumor xenograft model was constructed. Target genes and potential-binding sites were predicted using online databases and further confirmed via dual-luciferase reporter assay. Relative factors expressions were determined via quantitative real-time polymerase-chain reaction and Western blot as needed. LINC01315 was high-expressed in CRC and ALDH+ cells. LINC01315 silencing suppressed the migration, invasion, and sphere formation of CRC cells and tumor growth, and downregulated expressions of CSC molecules (ALDH, cluster of difference 44 (CD44), Prominin, and sex determining region Y-box 2 (SOX2)), Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Vimentin but upregulated E-Cadherin expression. MiR-484 could competitively bind with LINC01315, and LINC01315 silencing promoted miR-484 expression. The level of Delta Like Non-Canonical Notch Ligand 1 (DLK1), the target gene of miR-484, was enhanced by overexpressed LINC01315 yet was suppressed by LINC01315 silencing. Also, DLK1 silencing reversed the effects of downregulated miR-484 on migration, invasion, sphere formation, and CSC molecules expressions in CRC cells. LINC01315 silencing modulated CSC properties and epithelial-to-mesenchymal transition via miR-484/DLK1 axis.

已有研究发现长链非编码RNA（long non-coding RNA，lncRNA）基因间长链非编码RNA 01315（LINC01315）参与多种癌症的发生发展，但该分子在结直肠癌（colorectal cancer，CRC）中的具体作用与功能仍有待阐明。本研究通过生物信息学分析收集了结直肠癌组织中LINC01315的表达数据，并采用醛脱氢酶（aldehyde dehydrogenase，ALDH）检测联合流式细胞术（flow cytometry）分离结直肠癌干细胞（cancer stem cells，CSCs）。分别通过划痕实验、Transwell实验及成球实验（sphere-formation assay）检测转染后结直肠癌来源CSCs的迁移、侵袭及干细胞成球能力；构建异种移植瘤模型（tumor xenograft model）验证体内功能。通过在线数据库预测LINC01315的靶基因及潜在结合位点，并采用双荧光素酶报告基因实验（dual-luciferase reporter assay）进行验证；根据实验需求，采用实时定量聚合酶链反应及蛋白质印迹（Western blot）检测相关分子的表达水平。实验结果显示，LINC01315在结直肠癌组织及ALDH阳性（ALDH+）CSCs中呈高表达。沉默LINC01315可抑制结直肠癌细胞的迁移、侵袭及成球能力，并阻滞体内肿瘤生长；同时可下调CSCs相关分子（ALDH、簇分化抗原44（CD44）、CD133（Prominin）及性别决定区Y框蛋白2（SOX2））、锌指E盒结合同源框1（ZEB1）与波形蛋白（Vimentin）的表达，而上调E-钙粘蛋白（E-Cadherin）的表达水平。miR-484可与LINC01315竞争性结合，沉默LINC01315可促进miR-484的表达。Delta样非经典Notch配体1（Delta Like Non-Canonical Notch Ligand 1，DLK1）作为miR-484的靶基因，其表达水平可被过表达的LINC01315上调，而被LINC01315沉默所抑制。此外，沉默DLK1可逆转miR-484表达下调对结直肠癌细胞迁移、侵袭、成球能力及CSCs相关分子表达的影响。综上，沉默LINC01315可通过调控miR-484/DLK1轴，影响结直肠癌CSCs的干性特征及上皮间质转化进程。