| Acute myeloid leukemia(AML)is a kind of neoplasm that resulted from the malignant proliferation of myeloid progenitor cells in hematopoietic system.The AML constitutes about 20%of all acute leukemia incidences in children.The risk factors for acute leukemia include ionizing radiation,poisonous chemicals,virus infection,cytotoxic drugs,and genetic susceptibility.However,the molecular mechanism in acute leukemia pathogenesis is still obscure.Although new techniques such as stem cell transplantation have been applied,chemotherapy was still the major therapeutic measure in treatment of leukemia.A great deal of patients relieved after chemotherapy,but chemotherapy was also found to have no effect on quite a few leukemia patients.Additionally,leukemia has been found to recur among patients in complete remission.These failures in leukemia chemotherapy could be mainly attributed to multidrug resistance(MDR).Therefore,the investigation on molecular mechanism of MDR and increasing the sensitivity of leukemia cells to drugs that were applied in chemotherapy become one of the major objects in scientific and clinical researches on leukemia.The overexpression of p-glycoprotein(P-gp)that was encoded by multidrug resistance gene-1(mdr-1)on the surface of neoplasmic cells was reported to be the major cause for MDR.It was also found that MDR of neoplasmic cells was potentially associated with autophagy.Autophagy is a kind of mechanism that manipulates cell death,but was different from apoptosis or programmed cell death.Autophagy was involved in the regulation on development and the maintenance of metabolic balance.It also plays an important role in the pathogenesis of cancers.Cell autophagy is an evolutionary relatively conservative metabolic process.Basal autophagy levels are increased in a variety of tumor cells,which has a very important effect on cells.The increase of autophagy levels can degrade these dysfunctional and excess proteins or defective organelles,which play an important role in the recovery and reuse of proteins and amino acids,thereby promoting tumor cell survival.It's conducive to tumor cells to cope with oxidative stress or external injury.During chemotherapy,the continuous enhancing of autophagy levels can also promote drug efflux,which is helpful for tumor cells to produce toleration to chemotherapy drugs,promoting the production and progression of multidrug resistance in tumor cell.High mobility group box-1 protein(HMGB1)is a typical molecule that is associated with damage.It binds to certain transcription factors under normal conditions,but is released to extracellular spaces in case of stress.HMGB1 expression was increased in many leukemia cells including AML.HMGB1 affects leukemia cell apoptosis process by regulating Bcl-2 and Caspase-3.It was demonstrated by experiments that binding of HMGB1 and Bcl-2 led to the assembly of ophagosome in leukemia cells.Moreover,exogenous HMGB1 could induce autophagy,increasing the leukemia cell survival ability and resistance to oxidative stresses.In summary,HMGB1 could be potentially regarded as a target in treatment of leukemia.microRNA(miRNA)is a small endogenous non-coding RNA molecule that contains about 22 to 25 nucleotides.miRNAs function via base-pairing with complementary sequences of mRNAs,leading to the degeneration of targeting mRNA by RNA-induced silencing complex(RISC).miRNAs were found to be actively involved in pathogenesis of cancers that they could be oncogene or anti-oncogene.In the present study,we firstly detected the expression levels of miR-142-3p and HMGB1 in peripheral blood monouclear cells of 23 children with AML and 15 controls by qRT-PCR and Western blot,respectively.The results showed that miR-142-3p was significantly decreased and HMGB1 was increased in AMLs compared with controls.Moreover,the bioinformatics analysis revealed that HMGB1 is a potential target of miR-142-3p.To explore the effect of miR-142-3p on the sensitivity of HL60/ATRA and HL60/ADR to chemotherapeutic drugs by targeting HMGB1,we detected the expression of miR-142-3p and HMGB1 in HS-5,HL60,HL60/ATRA and HL60/ADR cells using qRT-PCR and Western blot,examined the effect of miR-142-3p and co-transfected miR-142-3p and HMGB1 on cell viability and apoptosis in HL60/ATRA,HL60/ADR cells,and then tested the relationship between miR-142-3p and HMGB1 by Dual-luciferase reporter system assay.Finally,the molecular mechanism by which miR-142-3p influences the sensitivity of HL60/ATRA and HL60/ADR cells to ATRA/ADR through targeting HMGB1 was investigated.In this study,we aimed to investigate the effect of miR-142-3p on multidrug resistant leukemic cell lines HL60/ADR and HL60/ATRA by targeting HMGB1 and the possible mechanism,which provided new ideas for the treatment of leukemia and the multidrug resistance in clinical practice.This research includes the following three parts:Part ?:Expression of miR-142-3p and HMGB1 in AML and its significance.Part ?:Effect of miR-142-3p on the drug sensitivity of AML cells by targeting HMGB1.Part ?:Molecular mechanism by which miR-142-3p regulates the drug sensitivity of AML cells through targeting HMGB1.Main Content:Part ?:Expression of miR-142-3p and HMGB1 in AML and its significanceMethods1.The differential expression of miR-142-3p and HMGB1 mRNA in peripheral blood monocytes of normal children and children with AML was detected by qRT-PCR.2.The expression of HMGB1 protein in peripheral blood monocytes of normal children and children with AML was detected by Western blot.3.The target gene of miR-142-3p was predicted by online bioinformatics softwares including TargetScan,Pictar,mirBase,microRNA.org.Results1.Compared with normal children,the expression level of miR-142-3p mRNA in peripheral blood mononuclear cells of children with AML was significantly decreased,and the expression level of HMGB1 mRNA and protein was significantly increased.2.Many bioinformatics softwares indicated that HMGB1 is a potential target of miR-142-3p.Part ?:Effect of miR-142-3p on the drug sensitivity of AML cells by targeting HMGB1Methods1.The drug resistance of HL60/ATRA and HL60/ADR cells was detected by MTT assay.2.The levels of miR-142-3p and HMGB1 in HS-5,HL60,HL60/ATRA,HL60/ADR cells were detected by qRT-PCR and Western blot.3.MTT and flow cytometry were used to detect the effect of miR-142-3p on cell viabity and apoptosis of HL60/ATRA,HL60/ADR cells treated with miR-142-3p and co-transfected miR-142-3p and HMGB14.Dual-luciferase reporter system assay was used to detect targeted relationship between miR-142-3p and HMGB1.5.qRT-PCR and Western blot were used to detect the effect of miR-142-3p on expression of HMGB1 mRNA and protein.Results1.Compared with AML cell HL60,the expression of miR-142-3p in HL60/ATRA and HL60/ADR was significantly decreased,while the expression of HMGB1 mRNA and protein was significantly increased.2.The results of fuction study showed that miR-142-3p overexpression couldinhibit the viability of HL60/ATRA and HL60/ADR cells,and promote its apoptosis,while HMGB1 overexpression could reverse the effect of miR-142-3p on HL60/ATRA and HL60/ADR cells.3.Dual-luciferase reporter system assay show that HMGB1 is a target gene ofmiR-142-3p.4.miR-142-3p overexpression and deletion could inhibit and induce HMGB1 expression,respectively.Part?:Molecular mechanism by which miR-142-3p regulates the drug sensitivity of AML cells through targeting HMGB1Methods1.The expression of P-gp protein in each group cells was detected by Western blot.2.The expression of autophagy-related protein in each group cells were detected by Western blot.Results1.The expression levels of P-gp and autophagy-related proteins in HL60/ADR and HL60/ATRA cells were significantly decreased than those in HL60 cells.2.The expression of P-gp and autophagy-related proteins in HL60/ATRA?HL60/ADR transfected si-HMGB1 were significantly inhibited.3.miR-142-3p overexpression could inhibit the expression of P-gp,Atg5 and LC3-?/LC3-? ratio,while HMGBI overexpression could reverse the effect of miR-142-3p on P-gp,Atg5 and LC3-?/LC3-? ratio in HL60/ATRA and HL60/ADR cells.Conclusion1.Compared with normal children,the expression level of miR-142-3p in children with AML was significantly decreased,while the expression level of HMGB1 was significantly increased.Bioinformatics softwares indicated that HMGB1 is a potential target of miR-142-3p.2.miR-142-3p could inhibit the viability of HL60/ATRA,HL60/ADR cells and promote its apoptosis to enhance the sensitivity of HL60/ATRA and HL60/ADR to ATRA/ADR through targeting HMGB1.3.miR-142-3p/HMGB1 enhanced the chemosensitivity of HL60/ATRA and HL60/ADR to ATRA/ADR by inhibiting the expression of P-gp and cell autophagy in HL60/ATRA and HL60/ADR cells. |
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