Department of Breast Oncology, Xiang Yang Central Hospital, The Affiliated Hospital of Hubei College of Arts and Sciences, 136 Jingzhou Street, Xiangyang, 441021, Hubei, China.
Disinfection Supply Center, The Second People's Hospital of Lianyungang, Lianyungang, 222023, China.
Hum Cell. 2020 Apr;33(2):386-396. doi: 10.1007/s13577-019-00301-0. Epub 2020 Feb 17.
Long non-coding RNAs (lncRNAs) play key roles in tumorigenesis. It has been reported that the lncRNA nuclear-enriched abundant transcript 1 (NEAT1) may act as an oncogenic regulator in several cancers. However, the biological mechanism of action of NEAT1, particularly the miRNA sponge role in colorectal cancer (CRC), has not been fully elucidated. In our study, the expression of NEAT1, miR-205-5p, and vascular endothelial growth factor A (VEGFA) in CRC cell lines were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Cell migration and invasion were examined by wound healing and transwell assays, respectively. RNA-binding protein immunoprecipitation (RIP), and dual-luciferase and RNA pull-down assays were conducted to determine the correlation between miR-205-5p and NEAT1 or VEGFA. VEGFA, matrix metalloproteinase (MMP)2, and MMP9 protein and mRNA expression were measured by western blotting and RT-qPCR analysis, respectively. Our results demonstrated high expression of NEAT1 and VEGFA and low expression of miR-205-5p in CRC cell lines. The RIP and dual-luciferase assays confirmed miR-205-5p as a target of NEAT1. In addition, VEGFA was identified as a direct target of miR-205-5p. Inhibition of NEAT1 or overexpression of miR-205-5p was able to repress VEGFA expression. Moreover, downregulation of NEAT1 and VEGFA inhibited cell proliferation, migration, and invasion. NEAT1 overexpression facilitated tumor growth by modulating miR-205-5p. Taken together, lncRNA NEAT1 was found to be upregulated in CRC cell lines, promoting CRC cell proliferation, migration, and invasion through regulating the miR-205-5p/VEGFA signaling pathway. These findings suggest that NEAT1 may be a promising biomarker in CRC diagnosis and treatment.
长链非编码 RNA（lncRNA）在肿瘤发生中发挥关键作用。据报道，核富集丰富转录物 1（NEAT1）lncRNA 可能在几种癌症中作为致癌调节剂发挥作用。然而，NEAT1 的生物学作用机制，特别是在结直肠癌（CRC）中的 miRNA 海绵作用，尚未完全阐明。在我们的研究中，通过逆转录定量聚合酶链反应（RT-qPCR）分析评估 CRC 细胞系中 NEAT1、miR-205-5p 和血管内皮生长因子 A（VEGFA）的表达。通过细胞计数试剂盒-8（CCK-8）测定检测细胞增殖。通过划痕愈合和 Transwell 测定分别检测细胞迁移和侵袭。进行 RNA 结合蛋白免疫沉淀（RIP）以及双荧光素酶和 RNA 下拉测定，以确定 miR-205-5p 与 NEAT1 或 VEGFA 之间的相关性。通过 Western blot 和 RT-qPCR 分析分别测量 VEGFA、基质金属蛋白酶（MMP）2 和 MMP9 蛋白和 mRNA 的表达。我们的结果表明 CRC 细胞系中 NEAT1 和 VEGFA 表达升高，miR-205-5p 表达降低。RIP 和双荧光素酶测定证实 miR-205-5p 是 NEAT1 的靶标。此外，VEGFA 被鉴定为 miR-205-5p 的直接靶标。抑制 NEAT1 或过表达 miR-205-5p 能够抑制 VEGFA 表达。此外，下调 NEAT1 和 VEGFA 抑制细胞增殖、迁移和侵袭。通过调节 miR-205-5p，NEAT1 过表达促进肿瘤生长。总之，lncRNA NEAT1 在 CRC 细胞系中上调，通过调节 miR-205-5p/VEGFA 信号通路促进 CRC 细胞增殖、迁移和侵袭。这些发现表明 NEAT1 可能是 CRC 诊断和治疗的有前途的生物标志物。
