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CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells
Naser Jafari1, Hyunju Kim1,2, Rackhyun Park1,2
1Markey Cancer Center, University of Kentucky, Lexington, Kentucky, United States of America.
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.
Insights
The study found that Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) is crucial for cancer cell proliferation and invasion. Removing NOX4 significantly reduced HeLa cell growth and their ability to invade tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein expression is linked to cancer progression and metastasis.
- The precise role of NOX4 in cancer cell proliferation and invasion remains incompletely understood.
Purpose of the Study:
- To investigate the cellular functions of NOX4 in cancer progression.
- To explore the impact of NOX4 deficiency on HeLa cell proliferation, invasion, and migration.
Main Methods:
- Generation of NOX4 knockout HeLa cell lines utilizing the CRISPR-Cas9 gene editing system.
- Confirmation of NOX4 knockout through T7 endonuclease 1 assays, DNA sequencing, and Western blotting.
- Analysis of cell proliferation, cell cycle distribution, invadopodium formation, invasive activity, focal adhesion dynamics, and cell migration.
Main Results:
- NOX4 knockout cell lines exhibited reduced cell proliferation, characterized by an increased sub-G1 cell population and a decreased S/G2/M population.
- NOX4 deficiency led to a significant reduction in invadopodium formation and invasive capabilities.
- Absence of NOX4 resulted in decreased focal adhesion and impaired cell migration in HeLa cells.
Conclusions:
- NOX4 plays a critical role in facilitating both cell proliferation and invasion in HeLa cells.
- Targeting NOX4 may represent a potential therapeutic strategy for inhibiting cancer progression and metastasis.
