Effect Of CRISPR/Cas9-generated AEG-1 Knockout On The Biological Behavior And Intracellular Metabolites Of U251 Cells

Purpose:CRISPR/Cas9 technology was employed to knock out AEG-1 gene,and the effects of AEG-1 gene on cell biological behaviors(including cell apoptosis and cycle distribution,cell invasion and metastasis capacity)in U251 cells were detected.To look for suitable biomarkers to monitor AEG-1 expression level and biological behavior of U251 Cells,the feature of metabolite variation in the knockout cell lines were detected by hydrogen proton nuclear magnetic resonance spectroscopy(1H-NMRS).Methods:Firstly,three sgRNAs targeting AEG-1(AEG-1-sgRNA-1,AEG-1-sgRNA-2,and AEG-1-sgRNA-3)were designed according to the CRISPR official website(https://zlab.bio/guide-design-resources).Then,three AEG-1 targeting sgRNA/Cas9 expression vectors(PX459-AEG-1-1,PX459-AEG-1-2,and PX459-AEG-1-3)were constructed and DNA sequencing was performed.Then the vectors were transfected into human glioma U251 cells,and three stable monoclonal AEG-1 knockout U251 cell line was established through puromycin screening.The activity of sgRNA was identified by TA cloning sequencing.The knockout efficiency of AEG-1 was estimated by Western blot.The apoptotic rate and cell cycle distribution were valued by flow cytometry.Transwell chamber and scratch assay were utilized to appraise the effects of AEG-1 knockout on the invasion and migration of tumor cells.Finally,1H-NMRS technique was used to measure the changes of cell metabolites in the knockout cell lines.Results:DNA sequencing showed that the sgRNA/Cas9 expression vectors(PX459-AEG-1-1,PX459-AEG-1-2,and PX459-AEG-1-3)targeting AEG-1 gene were successfully constructed,and the constructed vectors were consistent with the experimental design.After the transfection of the recombinant plasmid and the screen of purinomycin,the three stable monoclonal AEG-1 knockout U251 cell lines were established successfully.TA cloning sequencing confirmed that all the three groups of sgRNA were active.Western blot results revealed that the AEG-1 protein levels of the three groups were significantly reduced,and the highest AEG-1 knockout efficiency was up to 99%.Flow cytometry detected that the apoptosis rate of knockout cell lines was increased,and the highest apoptosis rate was 3.6 times that of the control group(U251 cells transfected with empty Cas9 plasmid).The cell cycle distribution showed that the proportion of cells blocked in the G2 phase in the knockout cell lines was increased,and the highest was 62%in the third group.Transwell chamber test and scratch test displayed that the metastatic ability of AEG-1 knockout cell lines was significantly reduced.1H-NMRS detected that the percentages of tCho/Cr and Lac/Cr in AEG-1 knockout cell lines decreased on diverse degrees.Correlation analysis found that,with the decline of the AEG-1 expression level,the apoptosis rate of the AEG-1-knockout cell lines increased while the metastatic capacities decreased,the relative content of tCho and Lac reduced as well.Conclusions:The deviations in AEG-1 expression level influence the apoptosis rate and metastasis capacity of U251 cell,which could be monitored by the 1H-NMRS metabolites ratio.The tCho/Cr ratio and Lac/Cr ratio positively correlated with the AEG-1 expression level and malignant cell behaviours.This research may provide potential biomarkers for accurate preoperative diagnosis and AEG-1-targeting treatment evaluation of glioma in vivo in the future.