Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology,school of Life Sciences, Hubei University, Wuhan, 430062, China.
Genecreate Biological Engineering Co., Ltd., National Bio-industry Base, Wuhan, 430071, Hubei, China.
Sci Rep. 2019 Feb 4;9(1):1399. doi: 10.1038/s41598-018-34904-8.
MicroRNAs (miRNAs) bind to the 3'-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer.
微小 RNA（miRNAs）以序列特异性的方式与靶 mRNA 的 3'-非翻译区结合，从而抑制基因翻译。人类 miR-26a 已经得到了广泛的研究，但靶转录本远未完全确定。我们首先利用 CRISPR-Cas9 系统在人宫颈癌 HeLa 细胞中生成 miR-26a 敲除系。miR26a 敲除系显示出细胞生长增加和增殖改变。我们利用顺序窗口采集所有理论质谱 (SWATH-MS) 的蛋白质组学技术来比较野生型和敲除系之间的蛋白质丰度，试图鉴定受 miR-26a 影响翻译的转录本。具有显著变化的蛋白质的功能分类揭示了它们在应激反应、增殖、定位、发育、信号转导等方面的功能。选择细胞周期/增殖信号通路中的几种蛋白质通过 Western blot 和平行反应监测 (PRM) 进行验证。这三种方法之间令人满意的一致性表明了 SWATH-MS 定量的可靠性。在计算预测的靶标中，一部分靶标被 miR-26a 直接调控，这通过荧光素酶测定和 Western blot 证实。这项研究在 HeLa 细胞中创建了 miR-26a 靶向转录本的清单，并为进一步探索 miR-26a 在人类癌症中的功能提供了基础知识。
