title: Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.
abstract: 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.
authors.affiliation: 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.
authors.forename: Wenying
authors.initials: W
authors.lastname: Huo
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Guannan
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN, USA.
authors.forename: Jinggang
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Xuan
authors.initials: X
authors.lastname: Ouyang
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Chuanhe
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Baojing
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Women's Health Educational System.
authors.forename: Peixin
authors.initials: P
authors.lastname: Dong
authors.affiliation: Henan Agricultural University.
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Gynecology, Hokkaido University School of Medicine, Hokkaido University, Sapporo, Japan.
authors.forename: Hidemichi
authors.initials: H
authors.lastname: Watari
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN, USA.
authors.forename: Edward
authors.initials: E
authors.lastname: Chaum
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Pfeffer
authors.affiliation: Department of Pathology and Laboratory Medicine; Center for Cancer Research.
authors.forename: Junming
authors.initials: J
authors.lastname: Yue
keywords: CRISPR/Cas9; EMT.; lentiviral vector; miR-21; ovarian cancer