MiR-652-5p Regulates Glycolysis In Acute T-lymphocytic Leukemia Cells And Its Mechanism

Objective: Acute T-lymphocytic leukemia(T-ALL)is a rare and aggressive subtype of acute lymphoblastic leukemia(ALL),accounting for approximately 20% of adult ALL cases and 10-15% of childhood ALL cases.There are many studies on the coding sequences and functions of key genes in the T-ALL process.However,in addition to coding sequences,non-coding regulatory elements of the genome,such as miRNAs,have yet to be studied in depth.The aim of this study was to explore new miRNAs regulators and their functions in the development of T-ALL.Methods: Differences in miRNAs expression between 13 pediatric T-ALL bone marrow mononuclear cells and 3 normal pediatric T cells were detected by whole transcriptome sequencing;analysis of GEO database and q PCR assay of patient samples to verify miR-652-5p expression in T-ALL.T-ALL cell lines with stable inhibition of miR-652-5p were constructed using lentiviral vector technology,and cell function was detected by CCK-8,PI,Annexin-V/7-AAD,reactive oxygen species(ROS);cellular glycolysis products pyruvate and lactate were detected by gas chromatography,and cellular ATP production was detected by ATP assay,extracellular acidification rate(ECAR),extracellular ATP assay was performed to detect the extracellular acidification rate(ECAR)and extracellular oxygen consumption(OCR).A mouse model of miR-652-5p T-ALL inhibition was constructed,and the development of T-ALL was dynamically detected by mouse live imaging,and the infiltration of T-ALL cells was detected by HE staining.Target Scan Human database was used to predict miR-652-5p target gene,q PCR and WB to verify the expression of target gene TIGAR,and dual luciferase reporter gene assay to detect miR-652-5p and TIGAR binding;TIGAR knockdown T-ALL cell line was constructed using retrovirus,and CCK-8,PI,and Annexin-V/7-AAD,ROS assay to detect the cell function after miR-652-5p intervention,and WB to detect TIGAR-related protein expression.Results: RNA-seq sequencing data of 13 T-ALL cases and 3controls were analyzed to identify differentially expressed miRNAs in T-ALL,validate the aberrantly expressed miRNA: miR-652-5p,analyze the GEO database(GSE23024,GSE89078),and q PCR the bone marrow samples of 9 T-ALL patients and 5 controls.To verify the high expression of miR-652-5p in T-ALL.We successfully constructed stable miR-652-5p T-ALL cell lines(Jurkat and Molt-4 cells)and found that miR-652-5p inhibition reduced cell proliferation,decreased the proportion of division cycles,increased apoptosis,and increased cellular ROS accumulation;and decreased pyruvate,lactate and ATP production,decreased extracellular acidification rate and increased extracellular oxygen consumption.Inhibition of miR-652-5p slowed leukemia progression in a T-ALL mouse model,and HE staining revealed reduced infiltration of T-ALL cells in liver and spleen tissues and increased mouse survival.target Scan Human software predicted and validated the target gene of miR-652-5p,TIGAR,and knocked down in Jurkat and Molt-4 cells.T-ALL cells showed unchanged proliferation,increased apoptosis,elevated pyruvate,lactate,ATP,and increased extracellular acidification rate after TIGAR expression,and WB assay revealed that TIGAR inhibited PFKFB3 protein expression.Conclusion: In T-ALL,miR-652-5p increases cellular glycolytic capacity and promotes apoptosis by targeting and inhibiting TIGAR gene expression,thus in promoting T-ALL cellular processes in vitro and in vivo.