SLC1A5 Gene Promotes AML Progression Through Energy Metabolism Mediated By MTOR Pathway

Purpose: ASCT2(encoding gene SLC1A5)is a neutral amino acid transporter with glutamine as the preferred substrate and Na + substitution.It can transport glutamine into cells to provide energy.It is highly expressed in many tissues and cancer.Many studies have found that knocking down the SLC1A5 gene can inhibit the growth of polymers.Instead,the small molecule inhibitor V-9302 is a competitive antagonist of transmembrane glutamine flux,which can selectively supplement the aminotransferase ASTC2.At present,its effect is in the preclinical test stage,V-9302 The concentration of 25μmol/L can cause most of the lung,intestine,and fibroids cell lines.Our study started with two parts,and gradually found that SLC1A5 is related to the occurrence of AML,and observe its role in the AML cell lines KG-1and HL-60.Mechanism: It is found that V-9302 can promote the decomposition of AML cell lines KG-1 and HL-60,and may be used as an insertion drug to replace clinical practice to improve the cure rate of AML and reduce the recurrence rate.Materials and methods: This study is mainly divided into two parts.The first part: first use the bioinformatics method to mine the original m RNA expression data of AML from the TCGA database,collect a total of 151 tumor specimens,collect a total of 337 corresponding normal specimens from the GTEX database,and use the normalize function of the limma package to perform chip correction.The wilcox test was used to explore the difference in the expression of the gene between the two,and the Kaplan-Meier method was used for survival analysis.P<0.05 was considered to be significant.Analyze the difference in SLC1A5 gene expression and the difference in survival analysis caused by the difference in gene expression.Collect bone marrow samples from normal people or patients with bone marrow non-malignant diseases as the control group,and collect bone marrow samples from newly treated or retreated patients with AML as the experimental group.By extracting RNA from the bone marrow samples,reverse transcription into c DNA,using q-PCR technology,To explore the differences in the expression of the SLC1A5 gene,and to compare the database analysis data to further confirm that the gene is related to the occurrence of AML.In the AML cell lines KG-1 and HL-60,lentiviral plasmids were used to construct SLC1A5 knockdown stable transgenic strains,and the genes were analyzed by CCK-8,flow cytometry,western blot and other techniques for the proliferation and metabolism of the two cell lines.Oxidative stress,apoptosis and other aspects,and explore its mechanism of action.The second part: Use the small molecule inhibitor V-9302 to observe its effect on the AML cell lines KG-1 and HL-60 through CCK-8,flow cytometry,q-PCR,western blot and other techniques.Results: Part one: Bioinformatics analysis found that SLC1A5 is highly expressed in AML patients compared with normal people,and the high expression of this gene is related to survival.Our clinical data analysis of AML patients confirmed this conclusion.In the SLC1A5 knockdown AML cell line KG-1 and HL-60 stable transgenic strains constructed using lentiviral plasmids,compared with the normal expression of SLC1A5 KG-1 and HL-60 cells,as time increases,The proliferative activity gradually decreased.After 48 h,the expression level of reactive oxygen species in KG-1 and HL-60 cells increased,while the expression of GSH in the cells decreased,and the level of oxidative stress increased,and increased autophagy could be clearly observed,eventually leading to cell apoptosis under the combined action Increased,the mitochondrial apoptotic pathway is activated,the generation of these phenomena and the inhibition of the m-TOR signaling pathway leads to a decrease in the level of intracellular energy metabolism and energy deficiency.Part two: After V-9302 acts on the AML cell lines HL-60 and KG-1,the cell viability is obviously inhibited and is dose-dependent.At the same time,V-9302 promotes the apoptosis of HL-60 and KG-1.After acting on cells,it can increase the expression levels of pro-apoptosis-related protein BAX and Caspase3 activation fragment cleaved-Caspase3,while the expression of anti-apoptosis-related protein BCL2 decreases,and the ratio of BCL2/BAX decreases,confirming that it may activate mitochondrial apoptosis.Death pathway,causing apoptosis of HL-60 and KG-1.Conclusion: The high expression of SLC1A5 is related to the progress of AML.Knockdown of SLC1A5 can inhibit the growth and proliferation of AML cells by inhibiting the decrease in the level of energy metabolism mediated by the mTOR signaling pathway;use a competitive transmembrane glutamine flux antagonist V-9302 can promote the apoptosis of AML cells and inhibit the progress of AML.