International Journal of Molecular and Cellular Medicine
مجله بین المللی سلولی و مولکولی
Int J Mol Cell Med
Medical Sciences
http://ijmcmed.org
1
admin
2251-9637
2251-9645
10.22088/IJMCM.BUMS
en
jalali
1401
8
1
gregorian
2022
11
1
11
2
online
1
fulltext
en
A Decrease in CD44 on Cell Surfaces (MKN-45 Cell Line) After RELA Knockout Using CRISPR/Cas9
Cancer
Cancer
Original Article
Original Article
<p><span style="font-family:Tahoma;"><span style="font-size:12px;"><span style="line-height:200%"><span style="unicode-bidi:embed"><span sans-serif=""><span style="line-height:200%"><span new="" roman="" times="">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 was significantly (P<0.001) decreased after CRISPR/Cas9 knockout. Compared to the control group, the MFI of CD44 in transfected cells was 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.</span></span></span></span></span><br>
</span></span></p>
RELA, knockout, CRISPR/Cas9, CD44, MKN-45 cell line
117
126
http://ijmcmed.org/browse.php?a_code=A-10-2492-3&slc_lang=en&sid=1
Saeid
Karimi
medicalgenetics.sk@gmail.com
100319475328460026148
100319475328460026148
No
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Sima
Salmani
siimasalmani@gmail.com
100319475328460026149
100319475328460026149
No
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Akram
Alizadeh
alizadehbio@gmail.com
100319475328460026150
100319475328460026150
No
Department of Tissue Engineering and Applied Cell Sciences, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Leila
Rezakhani
leila_rezakhani@yahoo.com
100319475328460026151
100319475328460026151
No
Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Zohreh
Saltanatpour
zohre_saltanatpour@yahoo.com
100319475328460026152
100319475328460026152
No
Pediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
Sorayya
Ghasemi
sorayya.ghasemi@gmail.com
100319475328460026153
100319475328460026153
Yes
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.