| Background & Objective: Acute myeloid leukemia(AML)is a highly heterogeneous group of hematological malignancies due to abnormal proliferation and differentiation of hematopoietic stem/progenitor cells,which is highly malignant and progresses rapidly.Most patients cannot be cured by chemotherapy regimens,and the 5-year overall survival rate is less than 50%.There is currently a great need to deeply explore the relevant biological mechanisms of AML in order to develop more effective diagnostic and therapeutic regimens.MicroRNAs(miRNAs),a class of short non coding single strandedRNAs,are involved in the regulation of target gene expression,and multiple pathways affect biological behaviors such as cell proliferation,differentiation,metabolism,senescence and death,and can promote or inhibit tumor cell growth.The exploration of the molecular mechanism provides a new direction for the development of new targeted therapeutics.In recent years,the discovery of novel regulated cell death processes,such as ferroptosis and autophagic death,has opened new avenues for the treatment of malignancies.Autophagy is a cellular activity process by which the body maintains homeostasis of the body environment through clearance of dead cells and other processes.Ferroptosis is a form of cell death that results from excessive intracellular iron accumulation accompanied by reactive oxygen species(ROS)imbalance,peroxidation of lipid structures in cell membranes,and depletion of glutathione(GSH).Studies have shown that autophagy plays a crucial role in the process of iron ion transport,and its balance point for the regulation of ferroptosis is a hot spot of current research.The role of miRNAs in the occurrence and development of AML has gradually attracted attention,but the mechanism research still needs to be further studied.Understanding the death forms and influencing factors of AML cells is of great significance for mining new prospects for AML treatment.This study aimed to explore the mechanism by which miR-20b-5p promotes AML cells to resist ferroptosis by regulating autophagy,and to provide a new strategy for the targeted therapy of AML.The role of miRNAs in the development and progression of AML has been gradually recognized,but the mechanism study still needs further insight,and understanding the death forms of AML cells and their influencing factors is of great significance to excavate new prospects for AML therapy.This study aimed to explore the related mechanisms by which miR-20b-5p participates in promoting AML cells to resist ferroptosis by regulating autophagy,providing a new strategy for targeted therapy of AML.Methods: 1.The expression of genes such as miR-20b-5p was analyzed in AML patients by screening the AML related miRNAs dataset with GEO database and combined TCGA database;Kaplan-Meier method was applied to perform the patient survival curve analysis associated with miR-20b-5p expression;Real time PCR(RT-PCR)experiments were used to validate the expression of miR-20b-5p in AML cells(THP-1,Kasumi-1,KG-1 and TF-1 cells)and AML patients,and the cell lines that underwent subsequent experiments were selected based on the results.2.Chi square test was applied to analyze the correlation between miR-20b-5p expression and clinical information such as age and gender of AML patients.3.CCK-8 cell proliferation assay was used to examine the activity changes of AML cells after inhibiting miR-20b-5p expression and/or adding cytarabine(Ara-c).4.The data were divided into two groups with high and low expression according to the expression of miR-20b-5p in AML patients in the TCGA database,the differential genes were matched to obtain,functional analysis was performed through KEGG as well as GO pathways,the related pathways of miR-20b-5p were clarified,and the analysis was verified by GSEA.5.The expression of LC3,an autophagic core protein,in AML cells after inhibition of miR-20b-5p expression or combination of Ara-c/Ara-c+ Idarubicin(IDA)treatment was determined by Western blot,immunofluorescence experiments,and transmission electron microscopy was used to determine the number of autophagosomes.6.Western blot assay was performed to detect the expression of mammalian target of rapamycin(m TOR),phospho m TOR in AML cells after inhibition of miR-20b-5p expression or combination with Ara-c.7.The expression of autophagy related genes Beclin1,ATG3,ATG4,ATG5,ATG7,ATG9,ATG10 and ATG12 in AML cells after inhibition of miR-20b-5p expression was determined byRT-PCR,Western blot was applied to detect the expression of Beclin1 protein after inhibition of miR-20b-5p,Pull-down assay and dual luciferase reporter assay were applied to verify the targeting of miR-20b-5p to Beclin1 gene.8.AML cells treated with Ferrostatin-1(Fer-1)to suppress miR-20b-5p expression or combined Ara-c/Ara-c+IDA were assessed for cell viability using CCK-8 assays.9.After inhibition of miR-20b-5p expression and/or combined Ara-c/Ara-c+IDA,calcein acetoxymethyl ester fluorescence probe was used to measure the expression levels of iron ions in AML cells;The lipid peroxidation kit was used to detect the expression level of malondialdehyde(MDA);The expression level of GSH was detected by micro reduced glutathione kit;The expression levels ofROS were detected by immunofluorescence,and the effects of miR-20b-5p and chemotherapeutic drugs on AML cells ferroptosis were clearly inhibited.10.AML cells treated with 3-Methyladenine(3-MA),an autophagy inhibitor,to suppress miR-20b-5p expression in combination with Ara-c,were assessed for levels of cellular iron,MDA,ROS and GSH expression to assess the effect of inhibiting autophagy on AML cells ferroptosis.Results: 1.The differentially expressed miRNAs datasets associated with AML patients were filtered out by GEO database,and combined with TCGA database,miR-20b-5p was found to be significantly upregulated in AML,and Kaplan-Meier survival analysis indicated that patients with high expression of miR-20b-5p had significantly worse survival than those with low expression;RT-PCR results showed that miR-20b-5p was highly expressed in AML cells(THP-1,Kasumi-1,KG-1,TF-1 cells)and AML patients,and the prognostic risk stratification of AML patients was associated with miR-20b-5p expression,and the expression of miR-20b-5p was significantly higher in poor prognosis group than in good prognosis and intermediate prognosis group.2.miR-20b-5p expression levels were not significantly associated with patient age,gender,number of naive leukocytes,normal karyotype,presence of FLT3-ITD mutation,NPM1 mutation,and CEBPA mutation.3.Inhibition of miR-20b-5p expression reduced THP-1,Kasumi-1 cell viability in a concentration dependent manner,and treatment with a combination of low-dose Ara-c for 48 h significantly potentiated the viability reduction in AML cells.4.KEGG,GO pathway analysis as well as GSEA analysis all indicated that miR-20b-5p was associated with cell autophagy.5.Western blot and fluorescence images showed that inhibition of miR-20b-5p expression or the combination of low-dose Ara-c/Ara-c+IDA significantly enhanced LC3 II protein expression in THP-1 and Kasumi-1 cells,and increased autophagosomes were observed by electron microscopy,which indicated that inhibition of miR-20b-5p expression and the combination of chemotherapeutic drugs could promote AML cells autophagy.6.The results of Western blot assay showed that inhibition of miR-20b-5p expression for 48 h,and inhibited miR-20b-5p expression for 24 h and 48 h in combination with Ara-c,significantly decreased p-m TOR protein expression levels in THP-1 and Kasumi-1 cells.7.The results ofRT-PCR and Western blot assay indicated that inhibition of miR-20b-5p expression could promote the expression of autophagy related gene Beclin1,and the results of Pull-down assay and dual luciferase reporter assay verified that miR-20b-5p could bind to Beclin1 targets and decrease the expression level of Beclin1.8.CCK-8 assay detected that the cell viability of THP-1,Kasumi-1 cells decreased after inhibition of miR-20b-5p expression or combined Ara-c/Ara-c+IDA,which was recovered after treatment with Fer-1.9.Inhibition of miR-20b-5p expression combined with Ara-c/Ara-c+IDA promoted iron accumulation,increased MDA andROS expression,and decreased GSH expression levels in THP-1 and Kasumi-1 cells.10.THP-1,Kasumi-1 cells with inhibition of miR-20b-5p expression combined with Ara-c were treated with 3-MA,the expression levels of cellular iron ions,MDA andROS were significantly decreased compared with control cells,and the expression level of GSH was back increased.Conclusions: 1.miR-20b-5p is highly expressed in AML patients and AML cells,and is positively correlated with poor prognosis.Inhibiting the expression of miR-20b-5p can reduce the activity of AML cells,and the combination of Ara-C can enhance the inhibition.2.Inhibiting the expression of miR-20b-5p can promote the autophagy of AML cells,and the combination of Ara-C/Ara-c+IDA can enhance the promoting effect.Inhibiting the expression of miR-20b-5p participates in the regulation of m TOR pathway and promotes the autophagy of AML cells.Inhibiting the expression of miR-20b-5p can specifically up regulate Beclin1 gene and promote the autophagy of AML cells.3.Inhibition of miR-20b-5p expression combined with Ara-c/Ara-c+IDA can promote iron enrichment and ferroptosis in AML cells.Inhibition of autophagy can reverse the ferroptosis promoted by inhibition of miR-20b-5p expression combined with Ara-C. |
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