Huo Wenying, Zhao Guannan, Yin Jinggang, Ouyang Xuan, Wang Yinan, Yang Chuanhe, Wang Baojing, Dong Peixin, Wang Zhixiang, Watari Hidemichi, Chaum Edward, Pfeffer Lawrence M, Yue Junming
Department of Pathology and Laboratory Medicine; Center for Cancer Research; Department of Physiology; Henan Agricultural University; Henan University of Animal Husbandry and Economy, P.R. China.
Department of Pathology and Laboratory Medicine; Center for Cancer Research.
J Cancer. 2017 Jan 1;8(1):57-64. doi: 10.7150/jca.16723. eCollection 2017.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.
CRISPR/Cas9（成簇规律间隔短回文重复序列）介导的基因组编辑是一种用于功能缺失研究的强大方法。在此，我们报告慢病毒CRISPR/Cas9载体在前体miRNA序列中引入突变的效率很高，从而导致miRNA表达和功能丧失。我们构建了四种靶向miR-21前体序列不同区域的慢病毒CRISPR/Cas9载体，并且发现这些慢病毒CRISPR/Cas9 miR-21 gRNA载体在前体序列中诱导了突变，DNA检测酶突变分析和桑格测序显示了这一点。选择两种miR-21慢病毒CRISPR/Cas9 gRNA载体来探究miR-21在卵巢癌细胞系SKOV3和OVCAR3中的功能。我们的数据表明，前体miR-21序列的破坏导致细胞增殖、迁移和侵袭减少。此外，CRISPR/Cas9介导的miR-21基因编辑使SKOV3和OVCAR3细胞对化疗药物治疗敏感。miR-21的破坏导致SKOV3和OVCAR3细胞中上皮-间质转化（EMT）受到抑制，这通过上皮细胞标志物E-钙黏蛋白的上调以及间质标志物基因波形蛋白和Snai2的下调得以证明。与对照相比，在用miR-21 gRNA转导的细胞中，miR-21靶基因PDCD4和SPRY2上调。我们的研究表明，慢病毒CRISPR/Cas9介导的miRNA基因编辑是解决miRNA功能的有效方法，并且miR-21的破坏抑制卵巢癌细胞中的EMT。
