Jafari Naser, Kim Hyunju, Park Rackhyun, Li Liqing, Jang Minsu, Morris Andrew J, Park Junsoo, Huang Cai
Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.
Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea.
PLoS One. 2017 Jan 18;12(1):e0170327. doi: 10.1371/journal.pone.0170327. eCollection 2017.
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.
NOX4蛋白表达增加与癌症进展和转移相关，但NOX4在细胞增殖和侵袭中的作用尚未完全明确。我们使用CRISPR-Cas9基因编辑系统构建了NOX4基因敲除的HeLa细胞系，以探究NOX4的细胞功能。转染CRISPR-Cas9构建体后，我们进行了T7核酸内切酶1分析和DNA测序，以产生并鉴定NOX4基因座的插入和缺失。我们通过蛋白质免疫印迹法确认了NOX4基因敲除。NOX4基因敲除细胞系的细胞增殖减少，亚G1期细胞群体增加，S/G2/M期细胞群体减少。此外，NOX4缺陷导致侵袭伪足形成和侵袭活性显著降低。另外，NOX4缺陷还导致HeLa细胞中粘着斑和细胞迁移减少。这些结果表明，NOX4是HeLa细胞有效增殖和侵袭所必需的。
