The AGS cell line was derived from fragments of a stomach tissue tumor resected from a 54-year-old caucasian female patient who had received no prior therapy. This cell line is closely related to gastric adenocarcinoma diseases. To date, several models of gastric carcinoma xenografts using different cell lines have been developed. AGS is one of the most common cell lines for the xenograft modeling of gastric cancer; however, there is limited information about the characteristics of this model. So researchers usually apply CRISPR/Cas9 technology to create gene knockout or gene knockin AGS cell lines.
AGS knockout cell line is clonally derived from the AGS cell line which has been transfected with an encoded vector, followed by stable cell selection. This cell line can stably express CRISPR Cas9 nuclease, GFP, and hygromycin resistance gene. In combination with separately transfected sgRNAs, the AGS-Cas9 KO cell line can be used to efficiently generate targeted genomic modifications including gene knockout, gene knockin, gene mutagenesis, gene tagging, etc. It is also an ideal cell line model for sgRNA screening and validation, either individually or in pools. So the AGS knockout cell line is a popular cell model in studies that involve Gastric cancer and diseases.
Knockout PDEF in AGS cells using CRISPR/Cas9 genome-editing system to study gastric cancer
Gastric cancer is one of the most common malignant tumors worldwide and has the second-highest incidence and mortality rate among malignant tumors in China. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors. Although PDEF plays an important role in tumorigenesis, its biological function in gastric cancer is still unclear. Researchers investigated PDEF expression in the gastric cancer cell line AGS and the normal gastric epithelial cell line GES; The CRISPR/Cas9 genome-editing system was used to knockout PDEF in AGS cells as a model for gastric cancer. were evaluated Then they used CCK-8, flow cytometry, scratch wound, and transwell assays to evaluate the cell proliferation, apoptosis, migration, and invasion of PDEF-knockout AGS cells respectively. The results illustrated that Knockout of the PDEF gene significantly inhibited the migration of AGS GC cells, which implied that PDEF plays an important role in the proliferation, migration, and invasion of AGS cells and may serve as a new treatment target in gastric cancer. The PDEF protein in the AGS cells transfected with the recombinant plasmidpX459-sgRNA1 was not expressed at all, within the sensitivity of the western blot assays. Knocked down PDEF expression, have significantly lowered ABS CRISPR cells’ ability of migration, invasiveness, and proliferative capacity.
CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation
LIM homeobox transcription factor 1, alpha (LMX1A) is downregulated in human gastric cancer (GC), functioning as a tumor suppressor. By sequencing analysis of LMX1A mRNA 3’-untranslated region(3’-UTR), it shows that microRNA-9(miR-9) putatively targets human LMX1A. In established gene-edited AGS cells and primary human GC cells, ectopic overexpression of miR-9 by a lentiviral construct decreased LMX1A 3’-UTR activity, causing LMX1A mRNA and protein downregulation. Functional analyses show that miR-9 overexpression enhanced GC cell survival and proliferation. On the contrary, miR-9 inhibition by antagomir-9 lentivirus increased LMX1A 3’-UTR activity to upregulate LMX1A mRNA and protein expression, causing GC cell apoptosis. CRISPR/Cas9-mediated LMX1A knockout promoted AGS cell survival and proliferation. Importantly, miR-9 and antagomiR-9 were ineffective in the function of LMX1A-knockout AGS cells. Therefore, the knockout AGS cell line is helpful in studying the affecting factors of gastric cancer.
CRISPR/Cas9 knockout AGS cell line strategies
Work flow of knockout AGS cell line
1. Zhang YQ, Pei JH, Shi SS, et al. CRISPR/Cas9-mediated knockout of the PDEF gene inhibits migration and invasion of human gastric cancer AGS cells. Biomed Pharmacother. 2019;111:76‐85.
2. Xiaohong Zhang, Yanqing Qian, Fan Li, Songhua Bei, Meiyi Li, Li Feng. microRNA-9 selectively targets LMX1A to promote gastric cancer cell progression. Biochemical and Biophysical Research Communications. 2018;405-412.