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Impact of NOX4 Knockout by CRISPR/Cas9 on the MCF-7, HCA-7 and UM-RC-6 Cancer Cells
Marzieh Javadi1, Hossein Sazegar1, Abbas Doosti2
1Department of Biology, Faculty of Science, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Background:
The second most common cause of mortality is cancer. Increased NOX4 expression is linked to cancer development and metastasis. However, the significance of NOX4 in cell growth and assault, remains unclear.
Objective:
This study aimed to evaluate the effect of NOX4 knockouts in MCF7, UM-RC-6, HCA-7 cell lines.
Materials And Methods:
The NOX4 gene was knocked out in MCF7, UM-RC-6, and HCA-7 cell lines through using CRISPR Cas-9 genetic engineering techniques. After transfection, the CRISPR Cas-9 cassette, the T7 endonuclease I, qPCR, and western blotting assay detected the NOX4 knockouts. MTT and Annexin assessed the percentage of cell proliferation and apoptosis. Real-time PCR was used to measure the expression of pro- and anti-apoptotic genes.
Results:
Occurrence of NOX4 gene knockout in the examined cell lines, was confirmed by q-PCR and Western blot (P<0.001). The NOX4-deleted cell lines with increased sub-G1 caused lowered cell proliferation and population at S / G2/ M phases. In Vitro, NOX4 silencing caused lowered expressions of anti-apoptosis genes BCL-2 and SURVIVIN (P<0.0001), leading to increased tendency of apoptosis in the cell lines (P<0.0001) of the apoptotic genes BAX, P53, FAS. Additionally, the MTT and Annexin results of the target gene NOX4 knockout inhibited proliferation, increased mortality rates (P<0.01), and increased apoptosis.
Conclusion:
The findings of this study indicate that using NOX4 as a target can have therapeutic value for creating potential treatments against breast, colorectal, and kidney cancers which shows a need for a deeper understanding of the biology of these cancers with direct clinical outcomes for developing novel treatment strategies.
Insights
Targeting the NOX4 gene through knockout significantly inhibited cancer cell proliferation and increased apoptosis. This suggests NOX4 is a potential therapeutic target for breast, colorectal, and kidney cancers.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Cancer is a leading cause of mortality worldwide.
- Increased NADPH Oxidase 4 (NOX4) expression is associated with cancer development and metastasis.
- The precise role of NOX4 in cancer cell growth and survival remains largely undefined.
Purpose of the Study:
- To investigate the functional role of NOX4 in cancer.
- To evaluate the impact of NOX4 gene knockout on cancer cell proliferation and apoptosis.
- To assess the therapeutic potential of targeting NOX4 in specific cancer cell lines.
Main Methods:
- Utilized CRISPR-Cas9 gene editing to achieve NOX4 knockout in MCF7, UM-RC-6, and HCA-7 cancer cell lines.
- Confirmed NOX4 gene disruption using quantitative PCR (qPCR) and Western blotting.
- Assessed cell proliferation and apoptosis rates via MTT assays and Annexin staining, respectively.
- Quantified the expression levels of key pro-apoptotic and anti-apoptotic genes using real-time PCR.
Main Results:
- Successful NOX4 gene knockout was confirmed across all tested cell lines (P<0.001).
- NOX4-deleted cells exhibited reduced proliferation, with a notable decrease in S/G2/M phase populations and increased sub-G1 phase.
- NOX4 silencing led to decreased expression of anti-apoptotic genes (BCL-2, SURVIVIN) and increased expression of pro-apoptotic genes (BAX, P53, FAS), significantly promoting apoptosis (P<0.0001).
- MTT and Annexin assays confirmed that NOX4 knockout inhibited proliferation, increased cell mortality (P<0.01), and induced apoptosis.
Conclusions:
- NOX4 plays a critical role in promoting cancer cell proliferation and inhibiting apoptosis.
- Targeting NOX4 presents a promising therapeutic strategy for cancers including breast, colorectal, and kidney cancer.
- Further research into NOX4's biological functions is warranted for developing novel cancer treatment strategies.
