State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China.
Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing, China.
Cell Prolif. 2018 Oct;51(5):e12489. doi: 10.1111/cpr.12489. Epub 2018 Jul 30.
Emerged evidence demonstrates that long non-coding RNAs (lncRNAs) may play quintessential regulatory roles in the cellular processes, tumourigenesis and the development of disease. Though focally amplified lncRNA on chromosome 1 (FAL1) has been identified to have crucial functions in many diseases, its biological mechanism in the development of Hirschsprung's disease (HSCR) still remains unknown.
The expression levels of FAL1 in HSCR aganglionic tissues and matched normal specimens were detected by quantitative real-time PCR (qRT-PCR). Cell proliferation and migration were detected by Cell Counting Kit-8 (CCK-8) assay, Ethynyl-deoxyuridine (EdU) assay and transwell assay relatively. Cell cycle and apoptosis were assessed using flow cytometer analysis. Moreover, the novel targets of FAL1 were confirmed with the help of bioinformatics analysis and dual-luciferase reporter assay. Western blot assay as well as RNA immunoprecipitation (RIP) assay was conducted to investigate the potential mechanism.
FAL1 expression was markedly down-regulated in HSCR aganglionic tissues and decreased FAL1 expression was associated with the diagnosis of HSCR. Cell functional analyses indicated that FAL1 overexpressing notably promoted cell proliferation and migration, while down-regulation of FAL1 suppressed cell proliferation and migration. Additionally, Flow cytometry assay demonstrated that knockdown of FAL1 induced markedly cell cycle stalled in the G0/G1 phase. Furthermore, FAL1 could positively regulate AKT1 expression by competitively binding to miR-637.
These results illuminated that FAL1 may work as a ceRNA to modulate AKT1 expression via competitively binding to miR-637 in HSCR, suggesting that it may be clinically valuable as a biomarker of HSCR.
有新的证据表明，长非编码 RNA（lncRNA）可能在细胞过程、肿瘤发生和疾病发展中发挥重要的调节作用。虽然在染色体 1 上局部扩增的 lncRNA（FAL1）已被确定在许多疾病中具有重要功能，但它在先天性巨结肠症（HSCR）发展中的生物学机制尚不清楚。
通过实时定量 PCR（qRT-PCR）检测 HSCR 无神经节细胞组织和匹配正常标本中 FAL1 的表达水平。通过细胞计数试剂盒-8（CCK-8）测定、Ethynyl-deoxyuridine（EdU）测定和 Transwell 测定相对检测细胞增殖和迁移。通过流式细胞仪分析评估细胞周期和细胞凋亡。此外，通过生物信息学分析和双荧光素酶报告基因测定证实了 FAL1 的新靶标。通过 Western blot 测定和 RNA 免疫沉淀（RIP）测定来研究潜在的机制。
FAL1 在 HSCR 无神经节细胞组织中表达明显下调，FAL1 表达降低与 HSCR 的诊断有关。细胞功能分析表明，FAL1 过表达显著促进细胞增殖和迁移，而 FAL1 的下调抑制细胞增殖和迁移。此外，流式细胞术分析表明，FAL1 的敲低导致细胞周期明显停滞在 G0/G1 期。此外，FAL1 可以通过竞争性结合 miR-637 正向调节 AKT1 的表达。
这些结果表明，FAL1 可能作为 ceRNA 通过竞争性结合 miR-637 来调节 HSCR 中的 AKT1 表达，表明它可能作为 HSCR 的生物标志物具有临床价值。
