Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.
Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.
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慢病毒CRISPR/CAS9载体介导的miR-21基因编辑抑制了卵巢癌细胞中间质转变的上皮。
DOI：
10.7150/jca.16723
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发表时间：
2017
影响因子：
3.9
通讯作者：
Yue J
中科院分区：
文献类型：
--
作者：
Huo W;Zhao G;Yin J;Ouyang X;Wang Y;Yang C;Wang B;Dong P;Wang Z;Watari H;Chaum E;Pfeffer LM;Yue J
CRISPR/Cas9(簇状规则间隔短回文重复序列)介导的基因组编辑是功能丧失研究的有力手段。在此，我们报道了慢病毒CRISPR/Cas9载体高效地在前体miRNA序列中引入突变，从而导致miRNA表达和功能的丧失。我们构建了四种针对miR-21前体序列不同区域的慢病毒CRISPR/Cas9载体，DNA测序和Sanger测序表明，这些慢病毒CRISPR/Cas9 miR-21 gRNA载体能够诱导前体序列发生突变。选择两个miR-21慢病毒CRISPR/Cas9 gRNA载体，检测miR-21在卵巢癌SKOV3和OVCAR3细胞中的功能。我们的数据表明，miR-21前序列的中断会导致细胞增殖、迁移和侵袭减少。此外，CRISPR/Cas9介导的miR-21基因编辑使SKOV3和OVCAR3细胞对化疗药物敏感。阻断miR-21导致SKOV3和OVCAR3细胞上皮细胞向间充质转化(EMT)的抑制，表现为上皮细胞标志物E-钙粘素上调和间质标志物基因Vimentin和SNAI2下调。与对照组相比，miR-21gRNAs转导的细胞中miR-21靶基因PDCD4和SPRY2表达上调。我们的研究表明，慢病毒CRISPR/Cas9介导的miRNA基因编辑是解决miRNA功能的有效途径，而miR-21的干扰抑制了卵巢癌细胞的EMT。
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.