| Objective:Acute leukemia is a kind of malignant clonal disease which is arising from hematopoietic stem progenitor cells in bone marrow with a high mortality.Acute myeloid leukemia(AML)is a common type leukemia in adults,accounting for 60%-70% of acute leukemia.So far,the treatment of AML is still largely dependent on combination chemotherapy.However,the resistance to chemotherapy drug of leukemia cells is the key point to refractory and replased leukemia,and is the most important cause of chemotherapy failure.miRNA is an evolutionarily high conserved group of endogenous noncoding RNA with 19-25 nucleotides in length.It is known that miRNA could regulate revelant gene expression at post-transcriptional level by pairing to the binding sites within 3'untranslated region of target mRNA.It is closely related with the sensitivity of tumor cells to chemotherapeutic drugs.miR-199a-5p is an important miRNA of human body.It is related to myocardial hypertrophy,bone development,tumor development and multidrug resistance(MDR).Studies have shown that the low expression of miR-199a-5p is associated with the resistance of hepatocellular carcinoma to chemotherapeutic drugs.Other studies have shown that the level of myocardial hypertrophy by rapamycin treatment is significantly reduced in miR-199a-5p transgenic mice.These studies suggest that miR-199a-5p plays an important role in the regulation of multidrug resistance and autophagy in tumor cells.Autophagy is a highly conserved life phenomenon widely exists in eukaryotic cells.It is a kind of improtant way to maintain a steady state of intracellular environment and genomic stability under stress conditions through autolysis of damaged proteins and organelles.Many studies have shown that the use of chemotherapy drugs to cancer cells can induce autophagy.Autophagy is involved in the generation and maintenance of MDR as a protective mechanism in most tumor cells.Studies have shown that there are significant differences in autophagy of bone marrow mononuclear cells among newly diagnosed acute leukemia patients,relapsed and refractory leukemia patients and complete remission leukemia patients,suggesting that autophagy may be involved in the regulation of acute leukemia multidrug resistance.In summary,in order to verify the regulation effect of miR-199a-5p and autophagy activity in AML drug resistance,this study take AML patients and AML sensitive and resistant cell lines as research objects,detect the activity of autophagy and the expression of miR-199a-5p,explore the regulation of autophagy and miR-199a-5p for leukemia MDR,verify its signaling pathway,and further confirm the functional target gene of miR-199a-5p to regulate autophagy and drugresistance.In this study,we clarify the role of miR-199a-5p in the drug resistance of acute myeloid leukemia,providing a new target for the treatment of multidrug resistant leukemia.Methods:1.Real time RT-PCR was used to detect the expression of miR-199a-5p in bone marrow mononuclear cells of 16 refractory/replased and 7 complete remission AML patients.2.Real time RT-PCR was used to detect the expression of miR-199a-5p in sensitive and resistant acute myeloid cell lines.3.K562/ADM and K562 cells were transfected with the mimic and inhibitor of miR-199a-5p using Lipofectamine 2000,real time RT-PCR was used to verify the transfection efficiency,and CCK-8 assay was used to detect the proliferation rate of cells.4.Westernblot was used to detect the expression of autophagy related protein LC3-II/I and Beclin1 in K562 and K562/ADM cells.5.Westernblot was used to detect the expression of LC3-II/I and Beclin1 in K562 and K562/ADM cells after 3-MA pretreatment.6.Combination of 3-MA and adriamycin were given to K562/ADM cells,CCK-8 assay was used to detect the proliferation rate of cells.7.K562/ADM and K562 cells were transfected with the mimic and inhibitor of miR-199a-5p using Lipofectamine 2000,westernblot was used to detect the expression of LC3-II/I,Beclin1 and P62 in K562 and K562/ADM cells.8.mGFP-RFP-LC3 method was used to observe the fluorescence of LC3 using the fluorescence microscope.9.Westernblot was used to detect the expression of phosphorylated and non phosphorylated AKT,mTOR,P70S6K1 protein.10.Bioinformatic was used to predict downstream target genes of miR-199a-5p in the regulation of autophagy.11.RT-PCR and Western blot were used to explore the expression of DRAM1 in K562 and K562/ADM cells.12.RT-PCR and Western blot were used to explore the expression of DRAM1 in K562 and K562/ADM cells when miR-199a-5p expression was changed by cell transfection.13.Double luciferase reporter gene test was used to verify the direct binding site of miR-199a-5p to DRAM1 3 'UTR region.14.Westernblot was used to detect the expression of LC3-II/I protein when DRAM1 was downregulated in K562/ADM cells.15.CCK-8 assay was used to detect the proliferation rate of cells when DRAM1 was downregulated in K562/ADM cells.Results:1.Real time RT-PCR was used to detect the expression of miR-199a-5p in bone marrow mononuclear cells of 16 relapsed and refractory AML patients(RR1-RR16)and 7 complete remission patients(CR1-CR7).The results showed that miR-199a-5p present a trend of lower expression compared with CR patients.2.Real time RT-PCR results showed that the expression of miR-199a-5p in K562/ADM and HL60/ADM was lower than the sensitive cells K562,HL60(P<0.05).3.miR-199a-5p transfection efficiency verification:in K562/ADM cells,miR-199a-5p mimic effectively enhanced the expression of miR-199a-5p compared with the NC mimic;in K562 cells,miR-199a-5p inhibitor effectively decreased the expression of miR-199a-5p compared with the NC inhibitor.CCK-8 test showed that cell proliferation activity of K562/ADM decreased significantly when treated with adriamycin after transfected by miR-199a-5p mimic;however,cell proliferation activity of K562 cells increased significantly when treated with adriamycin after transfected by miR-199a-5p inhibitor(P<0.05).4.Westernblot results showed that the expression of LC3-II/I and Beclin1 in K562/ADM were higher than in K562 cells under the basic state.Moreover,LC3-II/I and Beclin1 expression in both K562 and K562/ADM cells increased significantly after treated with different concentration of adriamycin.The effect is more significant and rapid in K562/ADM than in K562 cells(P<0.05).5.The expression of LC3-II/I and Beclin1 in the cells of K562 and K562/ADM after 3-MA pretreatment were lower than those of the untreated group(P<0.05).6.CCK8 assay results showed that the proliferation activity of K562/ADM cells was significantly decreased after 3-MA pretreatment(P<0.05).7.Westernblot showed that the expression of LC3-II/I and Beclin1 was increased and P62 was decreased after down-regulation of miR-199a-5p;meanwhile,the expression of LC3-II/I and Beclin1 was decreased and P62 was increased after miR-199a-5p was up-regulated(P<0.05).8.mGFP-RFP-LC3 test was used to detect the red and green fluorescence after transfection of miR-199a-5p mimic and inhibitor.After the down-regulation of miR-199a-5p in K562 cells,the number of yellow and red fluorescent particles increased,it indicated that autophagy was increased;when miR-199a-5p was up-regulated in K562/ADM cells,the number of yellow and red fluorescent particles decreased,so it means autophagy was inhibited.9.Westernblot showed that when miR-199a-5p expression was down regulated in K562 cells,p-AKT,p-mTOR,and p-P70S6K1 expression were up-regulated;when miR-199a-5p expression was up regulated in K562/ADM cells,p-AKT,p-mTOR,and p-P70S6K1 expression were down-regulated(P<0.05).10.Bioinformatics was used to predict DRAM1 as the downstream target gene of miR-199a-5p.11.Real time RT-PCR and westernblot results showed that the protein and mRNA of DRAM1 in K562/ADM cells was higher than those in K562 cells(P<0.05).12.Real time RT-PCR and westernblot results showed that DRAM1 mRNA and DRAM1 protein were decreased in miR-199a-5p mimic transfection group,and were increased in miR-199a-5p inhibitor transfection group(P<0.05).13.Double luciferase reporter gene test assay further confirmed that miR-199a-5p could significantly reduce the fluorescence intensity of DRAM1 wild-type 3 'UTR plasmid,but mutation of this binding site could block the occurrence of this phenomenon.14.LC3-II/I protein was obviously down-regulated in K562/ADM after the cell was transfected with DRAM1 siRNA(P<0.05).15.CCK-8 assay showed that K562/ADM cells become more sensitive to adriamycin when the expression of DRAM1 was decreased by siRNA(P<0.05).Conclusion:1.The expression of miR-199a-5p was significantly down regulated in acute myeloid leukemia resistant cell lines and bone marrow samples from patients with relapsed/refractory acute myeloid leukemia.miR-199a-5p expression was correlated with the sensitivity of AML cells to chemotherapeutic drugs.2.Compared with K562 cells,K562/ADM cells showed higher basic autophagy activity;after adriamycin treatment,the two groups of cells showed enhanced autophagy activity in response to environmental changes,autophagy activity enhancement of K562/ADM is more significant and rapid.3-MA inhibited autophagy of leukemia cells.The sensitivity of K562/ADM cells to chemotherapeutic drugs was enhanced after the inhibition of autophagy.3.The high expression of miR-199a-5p inhibited the autophagy acitvity of leukemia cells.The effect is due to inhibition of autophagy initiate rather than blocking the autophagy flow.PI3K/AKT/mTOR/P70S6K1 signaling pathway is involved in the regulation of miR-199a-5p on autophagy in AML drug-resistant cells.DRAM1 is a functional target gene for miR-199a-5p to regulate autophagy and drug resistance in acute myeloid leukemia. |
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