Study The Effects And Mechanisms Of IL-7 On Bone Marrow Mesenchymal Stromal Cells Promoting Drug Resistance Of Chronic Myeloid Leukemia Cells To TKIs

Background and objectives:Chronic myeloid leukemia(CML)is a myeloproliferative disease caused by the BCR/ABL fusion gene.Tyrosine kinase inhibitors(TKIs)imatinib and nilotinib,as molecularly targeted therapeutic agents,has been proved a remarker efficacy in the treatment of CML.However,15%-25% patients gradually become drug resistance to further treatment.Some patients of CML in chronic phase are primary resistant to TKIs.In the presence of mutations in the BCR-ABL kinase site,particularly in the T3151 mutation,40% of patients develop resistance to TKIs.The underlying mechanisms of these resistance are poorly understood.In recent years,a large number of studies have shown that bone marrow microenvironment plays an important role in the drug resistance of leukemia.Bone marrow mesenchymal stem cells is one of the most important cells in the microenvironment of bone marrow,which can differentiate into a variety of cells such as fat cells,fibroblasts,and they can secrete extracellular matrix proteins,cytokines and growth factors.IL-7 is one of the secreted cytokines,which can promote the synthesis of DNA in leukemia cells,and is closely related to the development of hematological malignancies.This study aims to investigate the expression of IL-7 in the bone marrow of patients with chronic myeloid leukemia,and research the effect and specific mechanism of IL-7 secreted by bone marrow mesenchymal stem cells in the resistance of chronic myeloid leukemia cells to TKIs.Methods:1.Collect the bone marrow of 24 cases of resistant patients of chronic myeloid leukemia(RCML),non resistant patients of chronic myeloid leukemia(SCML)and healthy people(NC).Detect the expression of IL-7 by using IL-7 ELISA kit with high sensitivity,and detect the expression of IL-7 gene in bone marrow mononuclear cells by fluorescence quantitative PCR.2.Isolate mesenchymal stem cells from resistant patients of chronic myeloid leukemia patients,non resistant patients of chronic myeloid leukemia and healthypeople,using Ficoll-Hypaque density-gradient centrifugation.ELISA method is used to detect the level of IL-7 in the medium of mesenchymal stem cells from resistant patients of chronic myeloid leukemia patients(RMSCs),non resistant patients of chronic myeloid leukemia(SMSCs)and healthy people(NCMSCs).3.The primary NCMSCs,SMSCs and RMSCs were used to simulate bone marrow microenvironment of healthy people and CML patients bone marrow microenvironment in vitro.K562 and BV173 cells were used to simulate CML leukemia cells.Leukemia cells and bone marrow mesenchymal stem cells were cocultured,constructing the regulation model between human bone marrow hematopoietic microenvironment and CML leukemia cell.4.NCMSCs,SMSCs and RMSCs were cocultured with K562/BV173 cells in different proportions,and the proliferation of leukemic cells was observed in different proportions.NCMSCs,SMSCs and RMSCs were cocultured with K562/BV173 cells for different time and the proliferation of leukemic cells was observed at different time points.The method of CCK8 was used to detect the proliferation of cocultured K562 and BV173 cells.The cell cycle was detected by flow cytometry.After imatinib and nilotinib were added to the coculture group,the method of AnnexinV/PI was used to detect cell apoptosis and their sensitivity to TKIs.The method of western blot was used to detect the related proteins of BCR/ABL,PI3K/AKT and JAK/STAT signaling pathway.5.Add IL-7 antibody or knockout IL-7 gene of RMSCs in RMSCs+K562 group.Cell proliferation,apoptosis and the expression of protein molecules of JAK/STAT signaling pathway were observed after imatinib and nilotinib treatment.6.NCMSCs,SMSCs and RMSCs and K562 cells were inoculated into BALB/c-nu nude mice to establish subcutaneous tumor model.The effect of three kinds of bone marrow mesenchymal stem cells(MSCs)on the proliferation,apoptosis,drug resistance of leukemia cells,and the expression of the related proteins of JAK/STAT signaling pathway was observed in vivo.Result:1.The primary NCMSCs,SMSCs and RMSCs cells were isolated and cultured.The three kinds of MSCs were similar,which were fibroblast-like spindle-shaped and showed a vortex-like growth.The immunophenotype of the cells obtained by flow cytometry was consistent with the characteristics of bone marrow mesenchymal stem cells.2.ELISA results showed that the IL-7 level in RCML patients was significantly higher than that in patients with SCML and NC.The level of IL-7 in RMSCs culture medium was significantly higher than the corresponding NCMSCs group and SMSCs group.3.The results of fluorescence quantitative PCR assay showed that the expression of IL-7mRNA in bone marrow mononuclear cells of RCML group was significantly higher.There was no significant difference between SCML group and NC group.4.Coculture experiments showed that the proliferation inhibition of MSCs cells was concentration-dependent.The low concentration of bone marrow mesenchymal stem cells had no significant effect on the proliferation of leukemia cells,but the high concentration of bone marrow mesenchymal stem cells can significantly inhibit the proliferation of leukemia cells,and the inhibitory effect was enhanced with the increase of the proportion of mesenchymal stem cells.The effect of MSCs on the proliferation of leukemic cells was time-dependent.With the extension of time,the inhibitory effect of MSCs on the proliferation of leukemic cells was enhanced.But there was no significant difference between three kinds of bone marrow mesenchymal stem cells on the proliferation of leukemia cells.5.Cell cycle test showed that with the extension of coculture time,the cell cycle was significantly blocked.The proportion of S phase decreased,while the proportion of G0/G1 cells increased.However,there was no significant difference between K562+NCMSCs,K562+SMSCs and K562+RMSCs three groups.6.After adding IM or NI,the results showed that with the increase of the concentration of IM or NI,the proliferation rate of leukemia cells gradually decreased,and the apoptosis rate increased gradually.The NCMSCs,SMSCs and RMSCs three groups showed the same trend of the intervention with IM or NI.However,at the same drug concentration,the cell proliferation rate of K562+RMSCs group was significantly was significantly higher than the control group,NCMSCs group andSMSCs group,but the rate of apoptosis was significantly lower than the control group,NCMSCs group and SMSCs group.After adding IL-7 neutralizing antibody and knocking out the RMSCs gene of IL-7 in the K562+RMSCs group,the proliferation rate decreased,while the apoptosis rate increased.These results indicated that RMSCs can promote the proliferation of K562 cells,inhibit the apoptosis of K562 cells,and played a protective role in the cells after treated with tyrosine kinase inhibitor.This protective effect was related to IL-7.7.The expression of related signal pathway proteins was detected by using western blot detection.The results showed that IM/NI resistance induced by RMSCs was independent of BCR/ABL,and the PI3K/AKT signaling pathway was not involved.IL-7/JAK1/STAT5 pathway mediated the drug resistance induced by RMSCs of CML cells to tyrosine kinase inhibitors.8.In subcutaneous tumor model of nude mice,the results showed that RMSCs could protect leukemic cells,decrease the apoptosis of cells and increase the survival of the cells,thus promoting the drug resistance,which is related to Caspase 3 and Bcl-2.Further detection of the related signaling pathway showed that the levels of pJAK1 and pSTAT5 protein in K562+RMSCs subcutaneous tumor tissue were significantly higher than those in other groups.After knocking out the RMSCs gene of IL-7,the expression level of pJAK1 and pSTAT5 protein in tumor tissue decreased,suggesting that the IL-7/JAK1/STAT5 signaling pathway was involved in the resistance of RMSCs to leukemia cells in vivo.Conclusion:In resistant patients of chronic myeloid leukemia,bone marrow mesenchymal stem cells secrete IL-7 abnormally which can activate JAK1/STAT5 signaling pathway,indepentdent BCR/ABL,thereby promoting its resistance to tyrosine kinase inhibitors.This study may provide a new target for the treatment of chronic myeloid leukemia.