Zhang Danhua, Zhou Jianping, Dong Ming
Department of General Surgery, Gastrointestinal Surgery, The First Hospital, China Medical University, Shenyang, 110001, China,
Dig Dis Sci. 2014 May;59(5):958-67. doi: 10.1007/s10620-013-2983-4. Epub 2013 Dec 27.
MicroRNAs (miRs) are small non-coding RNAs that play important roles in cancer development where they can act as oncogenes or as tumor-suppressors. MiR-34a is a tumor suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in colorectal cancer (CRC).
This study aims to show the dysregulation of miR-34a in CRC and to characterize the function and mechanism of miR-34a in CRC cell lines.
The expression of miR-34a was detected using real-time PCR on CRC tissues and adjacent non-tumorous tissues. The ELISA was used to assess vascular endothelial growth factor (VEGF). The focal adhesion kinase (FAK) and phosphorylated FAK Y397 (pY397FAK) were measured by Western blot. The functions of miR-34a in vivo were measured by migration, invasion, CCK-8 assay and flow cytometry.
MiR-34a was significantly downregulated and pY397FAK was upregulated in CRC cancer tissues. It plays an important role in inhibiting migration and invasion and in increasing apoptosis of CRC cells. Bioinformatic analysis suggested that VEGF may be a target of miR-34a, and this hypothesis was proved by ELISA and RT-PCR. The level of pY397FAK that could be activated by VEGF was downregulated in miR-34a overexpression CRC cell lines. The phosphorylation of FAK at 397 sites in miR-34a-stable cell lines was completely rescued by extra VEGF treatment.
MiR-34a is frequently downregulated in CRC and modulates the phosphorylation of FAK by negatively regulating VEGF.
微小RNA（miR）是一类小的非编码RNA，在癌症发展过程中发挥重要作用，它们既可以作为癌基因，也可以作为肿瘤抑制因子。MiR-34a是一种肿瘤抑制因子，在多种肿瘤类型中经常下调。然而，关于miR-34a在结直肠癌（CRC）中的作用知之甚少。
本研究旨在揭示miR-34a在结直肠癌中的失调情况，并阐明其在结直肠癌细胞系中的功能及机制。
采用实时PCR检测结直肠癌组织及相邻非肿瘤组织中miR-34a的表达。采用酶联免疫吸附测定（ELISA）评估血管内皮生长因子（VEGF）。通过蛋白质免疫印迹法检测粘着斑激酶（FAK）和磷酸化FAK Y397（pY397FAK）。通过迁移、侵袭、CCK-8测定和流式细胞术检测miR-34a在体内的功能。
结直肠癌组织中miR-34a显著下调，pY397FAK上调。它在抑制结直肠癌细胞迁移和侵袭以及增加细胞凋亡方面发挥重要作用。生物信息学分析表明VEGF可能是miR-34a的一个靶点，ELISA和逆转录-聚合酶链反应（RT-PCR）证实了这一假设。在miR-34a过表达的结直肠癌细胞系中，可被VEGF激活的pY397FAK水平下调。额外的VEGF处理可完全挽救miR-34a稳定细胞系中FAK在397位点的磷酸化。
miR-34a在结直肠癌中经常下调，并通过负调控VEGF调节FAK的磷酸化。
