Karimi Saeid, Salmani Sima, Alizadeh Akram, Rezakhani Leila, Saltanatpour Zohreh, Ghasemi Sorayya
Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Department of Tissue Engineering and Applied Cell Sciences, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Int J Mol Cell Med. 2022;11(2):117-126. doi: 10.22088/IJMCM.BUMS.11.2.117. Epub 2023 Mar 1.
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.
NF-κB信号通路被认为是胃肠道恶性肿瘤中由炎症诱导的致癌作用的关键通路。RelA转录因子是该信号通路的重要组成部分。此外，CD44与胃癌的肿瘤发生和转移有关。本研究的目的是检测RELA基因敲除对MKN45细胞中CD44表达的影响。采用CRISPR/Cas9技术在MKN-45细胞中敲除RELA。分析MKN45细胞中RELA敲除前后CD44的中位荧光强度（MFI）。CRISPR/Cas9载体pSpCas9(BB)-2A-Puro(PX459)用于gRNA克隆（两个引导序列）。对MKN-45细胞系进行共转染。用嘌呤霉素纯化共转染细胞，培养后通过实时PCR检测RELA基因表达。采用流式细胞术分析MKN45细胞中CD44+的MFI。结果显示，MKN45细胞中RELA基因外显子2和外显子3之间180个核苷酸序列缺失。CRISPR/Cas9敲除后，RELA表达显著降低（P<0.001）。与对照组相比，转染细胞中CD44的MFI显著降低（P<0.001）。RELA基因敲除显著降低了MKN45细胞中CD44的表达。可以得出结论，通过RelA的NF-κB信号通路与CD44表达相关，进而与胃癌的肿瘤发生相关。建议对此关系进行更多研究。
