J Cancer 2017; 8(1):57-64. doi:10.7150/jca.16723
Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells
1. Department of Pathology and Laboratory Medicine
2. Center for Cancer Research
3. Department of Physiology
4. Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN, USA
5. Henan Agricultural University
6. Henan University of Animal Husbandry and Economy, P.R. China
7. Department of Women's Health Educational System
8. Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, Sapporo, Japan.
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.
Keywords: miR-21, CRISPR/Cas9, lentiviral vector, ovarian cancer, EMT.
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. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells. J Cancer 2017; 8(1):57-64. doi:10.7150/jca.16723. Available from http://www.jcancer.org/v08p0057.htm