Reversal Of Glucocorticoid Resistance In Acute Lymphoblastic Leukemia Cells By MicroRNA-145

Objective: Acute lymphoblastic leukemia(ALL)is the most common malignant tumor in children.The event free survival(EFS)of ALL children reached 81 percent,but there is still 15-20% recurrence rate,and once the recurrence rate,the cure rate is less than 40%.Whereas glucocorticoids(GCs)resistance is one of the main causes of ALL recurrence.Micro RNAs(miRNA)is a small non-coding single strand of about 22 nucleotides RNA,which can participate in the regμlation of GCs sensitivity through many mechanisms and is closely related to ALL cell resistance.This study first analyzed the expression of miR-145 in ALL children and its effect on ALL prognosis,and then explored the effect of miR-145 on proliferation,apoptosis and autophagy of human T-ALL cell line CEM-C7 and CEM-C1 cells and whether it coμld reverse the resistance of drug-resistant CEM-C1 cells to dexamethasone(Dexamethasone,DEX),providing new ideas for exploring the target of drug resistance in children.Methods:1.Bioinformatics: using GEO database data sets to analyze the expression level of miR-145 in ALL children,and reveal the relationship between miR-145 and ALL children’s prognosis through TARGET database data.2.Cell Cμlture: incubation of dexamethasone resistant human ALL cell lines CEM-C1 cell lines and sensitive cell lines CEM-C7 using in vitro cell cμlture technology.3.Liposome Transfection: miR-145mimic、miR-145 mimic NC negative control and miR-145inhibitor、miR-145 inhibitor NC negative control were transfected into CEM-C1 cells by lipo-2000 mediated liposome transfection,and divided into MM、MMN、MI、MIN four groups.4.Cell Proliferation Inhibition Assay: CCK-8 kit was used to detect the inhibition of cell proliferation in four groups by different concentrations of DEX(20,40,80,160 and 320 mg/ml),thus revealing the role of miR-145 in CEM-C1 response to GCs.5.Acridine Orange Staining: exploring the apoptosis effect of miR-145 to CEM-C1.6.Flow Cytometry: analysis of the apoptosis effects of miR-145 to CEM-C1.7.Quantitative Real-Time PCR(qRT-PCR): to detect the expression of miR-145 in CEM-C1 and CEM-C7 cells,and the expression of apoptosis-related gene Bax and Bcl-2,autophagy-related gene LC3 and Beclin-1,resistance gene MDR1 in four groups of cells after transfection.8.Cell Immunoblot Experiment(Western Blot,WB): to to detect the expression of miR-145 in CEM-C1 and CEM-C7 cells,and the expression of apoptosis-related proteins Bax and Bcl-2,autophagy related proteins LC3 and Beclin-1、 resistance protein MDR1 in four groups of cells.9.3-methyladenine(3-MA)interferes with MM and MMN group cells,divided into MMN、MMN 3-MA、MM、MM 3-MA four groups of cells,and the expression of autophagy-related protein LC3,Beclin-1 and drug-resistant protein MDR1 were detected to further investigate the relationship between autophagy and GCS resistance of ALL cells after 24 h.Resμlts:1.Database resμlts showed that miR-145 expression levels in children with ALL decreased significantly(P<0.001).2.Prognosis of ALL children miR-145 high expression group was significantly better than that of low expression group(P<0.001).3.Compared with CEM-C7 cells,the expression of drug-resistant protein MDR1 was significantly increased in CEM-C1 cells.4.MiR-145 expression in CEM-C1 cells was significantly lower than that in CEM-C7 cells.5.The overexpression of miR-145 increased the inhibitory effect of GCS on the proliferation of CEM-C1 cells.6.Increasing the expression of miR-145 can improve the drug resistance of CEM-C1 cells and promote cell death by increasing the expression of pro-apoptotic gene/protein Bax,decreasing the expression of anti-apoptotic gene/protein Bcl-2 and drug resistance gene/protein MDR1.7.At the same time,increased autophagy gene/protein Beclin1 miR-145 and LC3-Ⅱinducing autophagy,thus reducing resistance gene/protein expression of MDR1.8.The expression of MDR1 in min+3-MA group increased after 3-ma intervention,and the expression of Beclin l in MM+3-MA group increased compared with MIN+3-MA,while the expression of MDR1 decreased.Conclusion:1.Low expression of miR-145 in ALL children suggests poor progn osis.2.MiR-145 can reverse glucocorticoid resistance by regμlating apopt osis and autophagy in CEM-C1 cells.