The Study Of Role And Mechanism Of MiR-96 And MiR-34a In Chronic Myeloid Leukemia

Part Ⅰ The mechanism of miR-96 via targeting the BCR-ABL1 fusion gene as a tumor suppressor in chronic myeloid leukemia progressionIntroduction:Chronic myelocytic leukemia(CML)is a malignant clonal myeloproliferative disorder that originates from bone marrow pluripotent hematopoietic stem cells.It is characterized by t(9;22)translocation to form BCR-ABL1 fusion gene,and the encoded BCR-ABL1 fusion protein plays an important role in the pathogenesis of CML.BCR-ABL1 fusion protein has tyrosine kinase activity,then tyrosine kinase inhibition agents(TKIs)such as Imatinib inhibit the CML cell proliferation by targeting suppression of the tyrosine kinase activity of the BCR-ABL1 protein,which become the first-line treatment for CML patients.However,TKIs are mainly effective in patients with chronic CML,and have no significant effect on patients in the accelerated phase and blast phase.Moreover,the BCR-ABL1 gene can be mutated to develop resistance to TKIs treatment,resulting in TKIs not completely preventing the progression of CML.Therefore,studies on the regulation mechanism of BCR-ABL1 gene expression and blocking the progression of CML by regulating BCR-ABL1 gene expression are of great significance.MicroRNAs(miRNAs)are endogenous non-coding small RNAs of about 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level by degrading target mRNA or repressing translation.Studies have shown that miRNAs are involved in many biological processes such as cell proliferation,differentiation,invasion,metastasis and malignant transformation in many malignant tumors including CML.The BCR-ABL1 fusion gene is the molecular basis for the development of CML and plays an important role in driving the progression of CML disease.Related studies on whether miRNA can regulate the expression of BCR-ABL1 and then block or reverse the progression of CML are rare.This study found that the expression of miR-96 in bone marrow specimens of CML patients was significantly reduced compared with the chronic phase,and miR-96 regulates BCR-ABL1 through CML cell experiments,which further affects the CML cell proliferation and differentiation in the progression of CML.Thus,intervention of miR-96 may have a potential therapeutic effect on CML blast by regulating BCR-ABL1 at the transcriptional level.Methods:1.RT-PCR was used to detect the expression of miR-96 in bone marrow cells of CML patients.We collected bone marrow samples from patients with chronic phase(CML-CP),blast crisis(CML-BC),and iron-deficiency anemia(IDA)or healthy individuals and then isolated mononuclear cells.Extract total RNAs and determine miR-96 expressions by RT-PCR.2.The CML cell lines K562 and MEG01 were used to transfect miR-96 mimics and inhibitors respectively and then examine the role and mechanism of miR-96 via targeting the BCR-ABL1 fusion gene inhibiting the progression of CML by RT-PCR,Western Blot,cell transfection,flow cytometry,luciferase assay and DNA methylation.(1)Transfecting miR-96 mimics and inhibitors with wild-type/mutant BCR-ABL13’UTR reporter plasmids into K562 and MEG01 cells respectively,then detecting whether miR-96 can binding with BCR-ABL1 3’UTR by luciferase activity,and Western Blot assay were used to detect the expression of BCR-ABL1 fusion protein to explore the regulation and mechanism of miR-96 on BCR-ABL1.(2)After transfecting miR-96 mimics and inhibitors,observe the proliferation of K562 and MEG01 cells by EdU method,and the effect of miR-96 on the CML cell proliferation was investigated.(3)PM A was used to induce K562 and MEG01 cells to differentiate into megakaryocytes,hemin induced K562 cells to differentiate into red blood cells.RT-PCR was used to detect miR-96 expression,and flow cytometry was used to detect the expression of differentiation surface markers CD61 and CD71 to explore the role of miR-96 in the CML cell differentiation.(4)K562 and MEG01 cells were treated with different concentrations of chidamide and decitabine.,and the expression of miR-96 was detected by RT-PCR.The effect of histone acetylation and DNA methylation status changes on miR-96 expression in CML were investigated.(5)K562 and MEG01 cells transfected with miR-96 mimics were treated with different concentrations of imatinib,and the proportion of viable cells was measured and calculated by CCK8 method to explore the synergistic effect of miR-96 up-regulation on imatinib killing CML-BC cells.Results:1.miR-96 is down-regulated in the CML emergency.The expression of miR-96 was detected in 38 patients with CML-CP,12 patients with CML-BC,and 22 patients with IDA or healthy individuals.The results showed that the expression of miR-96 was decreased in CML-CP compared with the control group(P<0.05).Compared with CML-CP,the expression level of miR-96 in CML-BC was further decreased(P<0.05)..In addition,miR-96 expression was also down-regulated in CML-BC cell lines compared to CML-CP patients(P<0.001).These results indicate that miR-96 is involved in the progression of CML disease.2.miR-96 inhibits translation of BCR-ABL1 by targeting the 3’ UTR region.TargetScan shows that miR-96 may bind not fully complement with the 3’ UTR region of BCR-ABL1.After transfection of miR-96 mimics into K562 and MEG01 cells,dual luciferase assay showed that the wild-type BCR-ABL1 3’UTR luciferase activity decreased with increasing miR-96 expression,but to BCR-ABL1 3’UTR The mutant plasmid had no effect.Similarly,inhibition of miR-96 resulted in an increase in the dual luciferase activity of the wild type BCR-ABL1 3’ UTR reporter plasmid,while the mutant plasmid did not increase the dual luciferase activity.These results indicate that miR-96 can bind to the 3’UTR of BCR-ABL1.At the same time,the results showed that miR-96 inhibited BCR-ABL1 protein expression,autophosphorylation(pTyr)and downstream STAT5 and MEK/ERK signalingpathway activities without regulating BCR-ABL1 mRNA expression.These results indicate that miR-96 regulates the expression and function of BCR-ABL1 by inhibiting BCR-ABL1 translation.3.Low expression of miR-96 promotes proliferation of CML cells.The miR-96 mimics were transfected into CML-BC cell lines K562 and MEG01,and cell proliferation was measured by EdU method.It was found that overexpression of miR-96 can significantly reduce the proliferation of CML cells(P<0.01).Inhibition GENT of miR-96 was transfected into CML-BC cells,and the results of EdU showed that the proliferation of K562 and MEG01 cells was significantly increased(P<0.01).These indicate that low expression of miR-96 promotes proliferation of CML-BC cells.4.miR-96 is involved in the differentiation of CML-BC cells.In the in vitro differentiation induction model,it was found that the expression of miR-96 was increased in CML-BC cells after differentiation into red blood cells,indicating that miR-96 may participate in CML-BC cell differentiation and play a role in the transition of CML from chronic phase to blast phase.5.chidamide and decitabine mediate the recovery of miR-96 expression in CML-BC.Histone acetylation and DNA methylation play an important role in the regulation of miRNA expression.Our study found that miR-96 expression was up-regulated in a concentration-dependent manner and down-regulated in BCR-ABL1 expression in K562 and MEG01 cells treated with the novel histone deacetylase inhibitor chidamide,suggesting that miR-96 is in CML-BC the decrease in expression can be at least partially restored by chidamide.In addition,we compared whole-genome methylation microarrays of bone marrow CD34+cells of 5 CML patients and 5 normal donor,and found that the two methylation sites of miR-96 promoter are abnormally hypermethylated.Decitabine treatment up-regulate the expression of miR-96 in CML-BC cells,indicating that the low expression of miR-96 in CML-BC is regulated by abnormally hypermethylation of DNA.6.Up-regulation of miR-96 expression increases the sensitivity of CML-BC cells to imatinib.Different concentrations of imatinib were added to K562 and MEG01 cells transfected with miR-96 mimics,and the proportion of viable cells was measured by CCK8 method.We found that up-regulation of miR-96 levels increased the killing effect of imatinib on CML-BC cells in a concentration-dependent manner.Conclusions:1.miR-96 is down-regulated in the CML emergency.2.miR-96 binds directly to the 3’UTR of BCR-ABL1,inhibiting its protein translation,autophosphorylation,and downstream STAT5 and MEK/ERK signaling pathway activity.3.Low expression of miR-96 can promote the proliferation of CML-BC cells and participate in cell differentiation.4.There are CpG islands and two abnormal hypermethylation sites in the miR-96 promoter region of CML patients.chidamide and decitabine may restore the low expression of miR-96 in CML-BC through epigenetic mechanisms.5.Up-regulation of miR-96 expression enhances the killing effect of imatinib on CML-BC cells.miR-96 may be a new target for CML-BC therapy,and miRNA-based combination therapy provides a new strategy for the treatment of CML-BC.Part Ⅱ miR-374a sensitizes chronic myeloid leukemia cells to homoharringtonine by targeting c-MYCIntroduction:Chronic myeloid leukemia contains a chronic phase,an accelerated phase,and a blast phase with increasing malignancy.Tyrosine kinase inhibitors(TKIs)targeted inhibit the tyrosine kinase activity of BCR-ABL,however,TKIs are mainly effective in patients with chronic CML,but not in patients with accelerated and blast phase.Patients in chronic phase will also develop drug resistance and relapse after long-term use of drugs,eventually entering the end-stage of jerk,and at this phase the TKIs are not effective,and the prognosis is extremely poor.Therefore,it is urgent to find new treatment methods.Homoharringtonine(HHT)is one of the anti-tumor alkaloids isolated from the Chinese genus Cephalotaxus.It is a cell cycle-specific drug that promotes tumor cell differentiation and apoptosis by inhibiting protein synthesis in eukaryotic cells to play its anti-tumor effect.The study found that HHT has significant efficacy in patients with CML treated with TKIs,including T315I mutations.Therefore,the Chinese CML guidelines and NCCN guidelines recommend HHT as a treatment option for patients with TKIs resistance.However,the exact mechanism of HHT in the treatment of CML is not fully understood,and its use as a chemotherapy drug has toxic side effects such as heart and bone marrow suppression,which limits its wide application.Therefore,finding HHT sensitizer and increasing HHT drug sensitivity to reduce the dose of HHT are of great clinical significance.Studies have found that miRNA expression abnormalities are involved in the sensitivity of CML cells to chemotherapeutic drugs.For example,miR-124-3p increases the sensitivity of CML cells to chemotherapy through the miR-124-3p/B4GALT1 axis;up-regulation of miR-424 inhibits BCR-ABL activity and increased sensitivity to imatinib in CML cells;miR-486 regulates drug sensitivity of CML progenitor cells.MYC is an important proto-oncogene,which is involved in nearly half of human tumors and is closely related to malignant transformation and tumor progression.It is found that abnormally high expression of MYC plays an important role in TKI resistance and CML-BC cells.miRNA regulation of MYC plays an important role in a variety of disease processes,such as dilated cardiomyopathy,breast cancer,prostate cancer,liver cancer and the like.Therefore,we focused our research on whether miRNAs can regulate MYC pathways to increase the sensitivity of CML cells to HHT therapy.In this study,we found that the expression of miR-374a in bone marrow specimens of CML patients is low,especially in the blast phase.In CML cell line,miR-374a can regulate the expression of c-MYC to affect the proliferation and apoptosis of CML cells and promote the sensitivity of CML cells to HHT.In addition,HHT can up-regulate miR-374a expression in a time-and dose-dependent manner.Re-expression of miR-374a enhances the sensitivity of CML-BC cells to HHT and promotes the apoptosis of CML-BC cells,which forms the miR-374a-c-MYC-HHT loop to exert anti-tumor effects,and is expected to provide new ideas for intervention in CML blast.Methods:1.RT-PCR was used to detect the expression of miR-374a in bone marrow cells of CML patients.We collected bone marrow samples from patients with CML chronic and blast phase and control group and then isolated mononuclear cells.Extract total RNAs and determine miR-374a expressions by RT-PCR.2.Using the CML cell line K562 as a research object,transfected miR-374a mimics and inhibitors respectively,and studied the role and mechanism of miR-374a in synergy with HHT by targeting c-MYC gene inhibiting the progression of CML by RT-PCR,Western Blot,cell transfection,flow cytometry and other methods.(1)Flow cytometry was used to detect the apoptosis of K562 cells transfected with miR-374a mimetic and/or HHT,and the apoptosis of CML-BC cells induced by miR-3 74a in synergy with HHT was detected.(2)Flow cytometry was used to detect the apoptosis of K562 cells after transfection of miR-374a mimics and inhibitors,and to investigate the effect of miR-374a on apoptosis of K562 cells.Detect the apoptosis of K562 cells transfected with both miR-374a inhibitor and c-MYC overexpression plasmid and K562 cells transfected with both miR-374a mimics and c-MYC siRNA,and the role of c-MYC in miR-374a-induced apoptosis of K562 cells was investigated.(3)K562 cells were treated with HHT according to concentration gradient and time gradient.The expression of miR-374a and c-MYC were detected by RT-PCR.The expression of c-MYC protein was detected by Western Blot.The apoptosis of K562 cells was detected by flow cytometry.The effect of HHT on the expression of miR-374a and c-MYC in CML,and the mechanism of action of HHT,miR-374a and c-MYC in K562 cell apoptosis.Results:1.The expression of miR-374a in mononuclear cells of bone marrow specimens of CML patients is down-regulated,especially in the blast phase.2.Up-regulation of miR-374a expression enhances the sensitivity of CML-BC cells to HHT.Compared with the control group,miR-374a mimics alone could induce apoptosis of K562 cells.Compared with miR-374a mimics alone,transfection of miR-374a mimics plus HHT induced apoptosis of K562 cells more significantly(P<0.05),indicatingthat miR-374a mimics can enhance HHT-induced apoptosis more strongly(P<0.05),so up-regulation of miR-374a expression enhances K562 cell sensitivity to HHT.Compared with miR-374a mimics alone,the expression of miR-374a was higher after HTT addition(P<0.01),indicating that HTT also affected the expression of miR-3 74a in K562 cells.3.Down-regulation of c-MYC expression is one of the reasons for the up-regulation of miR-374a expression to increase the sensitivity of CML-BC cells to HHT.Bioinformatics analysis indicated that miR-374a may directly bind to the 3’UTR of c-MYC,which is an important oncogene during the occurrence and development ofCML,and c-MYC may be one of the targets of miR-374a to play an anti-leukemia role.Compared with the control group,miR-374a expression was up-regulated(P<0.01),while c-MYC mRNA level and protein level expression were down-regulated(P<0.05)after transfection of miR-374a mimics.Compared with miR-374a mimics alone,the expression of c-MYC in miR-374a mimics plus HHT group was significantly lower(P<0.05).On the contrary,compared with the control group,miR-374a expression was down-regulated after transfection with miR-374a inhibitor(P<0.01),and c-MYC mRNA level and protein level expression were up-regulated(P<0.05).The miR-374a mimics and c-MYC siRNA were transfected into K562 cells,and it was found that up-regulation of miR-374a expression and down-regulation of c-MYC expression induced apoptosis(P<0.05).In contrast,miR-374a inhibitor and c-MYC overexpression plasmids were transfected into K562 cells,and apoptosis was inhibited(P<0.05;P<0.05).Responsive experiments showed that miR-374a mimics-induced apoptosis was partially reversed after c-MYC overexpression.4.HHT mediate recovery of miR-374a expression in CML.K562 was incubated for 72 h and 96 h according to the HHT concentration gradient and the action time gradient.The results showed that the expression of miR-374a showed a dose-and time-dependent increase in HHT(P<0.01),and the expression of c-MYC mRNA and protein showed HHT dose-and time-dependent down-regulation(P<0.01).These results demonstrate that HHT is able to mediate the recovery of miR-374a expression in CML-BC.Conclusions:1.The expression of miR-374a in CML-CP patients was significantly lower than that in the control group.The expression of miR-374a was lower in CML-BC patients than in CML-CP.Up-regulation of miR-374a expression inhibited the proliferation of CML-BC cells and promoted CML-BC cell apoptosis.Up-regulation of miR-374a in combination with HHT increased apoptosis in CML-BC cells and increased sensitivity of CML-BC cells to HHT.2.miR-374a may increase the sensitivity of CML-BC cells to HHT through c-MYC mediation.That is,the anti-leukemia effect is exerted through the miR-374a-c-MYC pathway.3.HHT can up-regulate miR-374a expression in a time-and dose-dependent manner.Re-expression of miR-374a enhances the sensitivity of CML-BC cells to HHT and promotes apoptosis of CML-BC cells,thereby forming the miR-374a-c-MYC-HHT loop to exert an anti-tumor effect.The combination of miRNA and HHT may become a new strategy for CML treatment.