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Published on: June 6, 2020
A Decrease in CD44 on Cell Surfaces (MKN-45 cell line) After RELA Knockout Using CRISPR/Cas9
Saeid Karimi1, Sima Salmani1, Akram Alizadeh2
1Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Abstract:
The NF-kB signaling pathway was introduced as a key pathway in carcinogenesis that is induced by inflammation in gastrointestinal malignancies. The RelA transcription factor is an important component of this signaling pathway. Furthermore, CD44 is implicated in the tumorigenesis and metastasis of gastric cancer. The aim of this study was to assay the effect of RELA knockout on CD44 expression in MKN45 cells. CRISPR/Cas9 was used to knock out RELA in MKN-45. The median fluorescence intensity (MFI) of CD44 before and after RELA knockout is analyzed in MKN45. The CRISPR/Cas9 vector pSpCas9 (BB)-2A-Puro (PX459) was used for gRNA cloning (two guides). The MKN-45 cell line was co-transfected. The purified co-transfected cells with puromycin were cultured and used for the RELA gene expression assay by real-time PCR. Flow cytometry was used for the analysis of the MFI of CD44+ in MKN45. The results showed that 180 nucleotide sequences between exon 2 and exon 3 of RELA were deleted in MKN45. RELA expression significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells significantly decreased (P <0.001). Knockout of RELA significantly decreased CD44 expression in MKN45 cells. It can be concluded that the NF-kB signaling pathway via RELA is related to CD44 expression and consequently the tumorigenesis of gastric cancer. More studies about this relationship are recommended.
Insights
Knocking out the RELA gene significantly reduced CD44 expression in gastric cancer cells. This finding suggests a link between the NF-kB pathway, RELA, and CD44 in gastric cancer development.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- The Nuclear Factor kappa B (NF-kB) signaling pathway is crucial in inflammation-induced carcinogenesis, particularly in gastrointestinal cancers.
- The RelA transcription factor is a key component of the NF-kB pathway.
- CD44 is recognized for its role in the tumorigenesis and metastasis of gastric cancer.
Purpose of the Study:
- To investigate the effect of RELA gene knockout on CD44 expression in MKN45 gastric cancer cells.
- To explore the relationship between the NF-kB signaling pathway, RELA, and CD44 expression in gastric cancer.
Main Methods:
- CRISPR/Cas9 gene editing technology was employed to achieve RELA knockout in the MKN45 cell line.
- Real-time PCR was used to confirm RELA gene expression levels post-knockout.
- Flow cytometry was utilized to analyze CD44 expression by measuring median fluorescence intensity (MFI).
Main Results:
- CRISPR/Cas9-mediated knockout resulted in a significant deletion of 180 nucleotides in the RELA gene and a significant decrease in RELA expression (P<0.001).
- A significant reduction in CD44 expression, indicated by decreased MFI, was observed in MKN45 cells following RELA knockout compared to control groups (P <0.001).
- RELA knockout demonstrated a significant inhibitory effect on CD44 expression levels.
Conclusions:
- The NF-kB signaling pathway, specifically through the RELA transcription factor, is implicated in the regulation of CD44 expression in gastric cancer.
- These findings highlight a potential molecular link between RELA, CD44, and gastric cancer tumorigenesis.
- Further research into this relationship is recommended to elucidate its full implications for gastric cancer progression and potential therapeutic strategies.
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