title: CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.
abstract: Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Naser
authors.initials: N
authors.lastname: Jafari
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.|Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
authors.forename: Hyunju
authors.initials: H
authors.lastname: Kim
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.|Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
authors.forename: Rackhyun
authors.initials: R
authors.lastname: Park
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Liqing
authors.initials: L
authors.lastname: Li
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.|Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
authors.forename: Minsu
authors.initials: M
authors.lastname: Jang
authors.affiliation: Division of Cardiovascular Medicine, Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Morris
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.|Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
authors.forename: Junsoo
authors.identifier: http://orcid.org/0000-0002-2355-6760
authors.initials: J
authors.lastname: Park
authors.affiliation: Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.
authors.forename: Cai
authors.initials: C
authors.lastname: Huang
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics*; D049109:Cell Proliferation / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D022001:Focal Adhesions / Q000235:genetics; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics*; D009361:Neoplasm Invasiveness / Q000235:genetics; D009369:Neoplasms / Q000473:pathology*; D000069261:Podosomes / Q000235:genetics
chemical_list: D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human