A Decrease in CD44 on Cell Surfaces (MKN-45 cell line) After RELAKnockout Using CRISPR/Cas9

Document Type : Original Article

Authors
Saeid Karimi 1
Sima Salmani 2
Akram Alizadeh 3
Leila Rezakhani 4
Zohreh Saltanatpour 5 , 6
Sorayya Ghasemi 2
1 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
2 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
3 Department of Tissue Engineering and Applied Cell Sciences, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
4 Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
5 Pediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
6 Stem Cell and Regenerative Medicine Center of Excellence, Tehran University of Medical Sciences, Tehran, Iran.
10.22088/IJMCM.BUMS.11.2.117
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
Keywords
RELA
knockout
CRISPR/Cas9
CD44
MKN-45 cell line