| Background: Acute myeloid leukemia(AML)is a common malignant disease of the blood system that threatens human health.With high cost and long treatment cycle,AML causes great burden on patients and their families.Despite the emergence of new drugs and the improvement of chemotherapy regimens and treatment methods,the long-term prognosis of AML has not been significantly improved except for acute promyelocytic leukemia(APL).In the course of clinical treatment,most AML patients are prone to drug resistance,recurrence,loss of sensitivity to chemotherapy drugs,or even primary drug resistance,which is one of the reasons leading to poor long-term efficacy of AML.Therefore,it has become a research hotspot to deeply study the mechanism of AML resistance,search for new tumor therapeutic targets,and solve the problem of AML resistance.In recent years,it has been found that the interaction between tumor cells and tumor microenvironment is one of the important mechanisms for drug resistance of tumor cells(including AML).Bone marrow mesenchymal stem cells(BMMSCs)are important component in the bone marrow microenvironment of AML.They play an important role in protecting AML cells from apoptosis induced by chemotherapy drugs.However,the mechanism of its protection has not been fully elucidated.It has been found that in solid tumors,tumor cells recruit BMMSCs by secreting cytokines or chemokines,and culturing BMMSCs to become cancer ? associated fibroblasts(CAFs)supports the progression of malignant cells,including tumorigenesis,drug resistance,invasion and metastasis,immune escape and regulation of tumor inflammatory microenvironment.Fibroblast activation protein alpha(FAP α)is considered to be an important functional molecular marker of CAFs.Studies have shown that FAP α plays an important role in CAFs mediated tumor drug resistance,development and immune escape,and is related to the poor prognosis of tumor,becoming the target of tumor treatment.Since BMMSCs can be chemotactic into the solid tumor microenvironment,activated into CAFs,and participate in the formation of CAFs,can BMMSCs be activated into CAFs in blood tumor microenvironment? What role does FAP α play in mediating BMMSCs to promote AML anti-chemotherapy-induced apoptosis,and what is the mechanism? Can FAP α become a new target of AML drug resistance? Based on the above research background and the existing problems,we try to conduct related research.Objective: To investigate the expression level and clinical significance of FAP α in BMMSCs of AML patients,and the difference of FAP α expression was compared between BMMSCs in AML patients and healthy controls.The role and mechanism of FAP α in mediating BMMSCs to promote AML against Ara-C-induced apoptosis.Methods: BMMNCs from newly treated AML patients and healthy controls were obtained by isolation and purification.BMMSCs were obtained by in vitro subculture,and molecular markers on the surface of BMMSCs were identified by flow cytometry.The expression level of FAP α in BMMSCs was detected by flow cytometry,quantitative PCR and immunofluorescence.Evaluate the expression of FAP α in BM biopsy samples via immunohistochemistry,as well as to identify the correlation between the expression of FAP α in BM with clinical parameters and survival of newly diagnosed patients with AML.Finally,FAP α of BMMSCs was knocked down by small interference RNA.Flow cytometry was used to detect the changes in the apoptotic proportion of Kasumi-1 cells induced by cytarabine(Ara-C),and the potential mechanism was investigated by Western Blot.Results: 1.The experimental results demonstrated that cultured BMMSCs adhered to the culture flask,showing viscoplastic and fibroblast-like morphology.BMMSCs molecular markers were detected by flow cytometry after passage to the second generation,and CD14,CD34 and CD45 were low expression,while CD90 and CD105 were high expression;2.The results showed that the expression level of FAP α in BMMSCs of newly diagnosed AML patients was significantly increased than that of healthy control group,and the difference was statistically significant.Expression of FAP α in BMMSCs detected via FCM:(70.92 ± 4.38% vs 36.74 ± 10.37%;P=0.0072),and RTq PCR:(24.75 ± 2.75 vs.9.77 ± 1.94;P = 0.0001);3.Positive expression of FAP α was observed in the brown-yellow region of bone marrow stroma.The OD/Area value of FAP α in AML bone marrow biopsy was significantly higher than that in healthy control group(11.88 ± 4.55 vs.5.16 ± 3.67;P=0.0001;n=15).The OD/Area value of FAP α was positively correlated with the proportion of BM blasts detected by flow cytometry(r=0.878;P<0.0001).In 8 patients who did not receive chemotherapy,the OD/Area value of FAP α was negatively correlated with survival time(r=0.815;P=0.0137);4.The relative expression of FAP α m RNA in the FAP α?si RNA group was significantly lower compared with that in negative control(NC)?si RNA group(0.11 ± 0.01 vs.0.84 ± 0.08;P=0.0001);It suggested that si RNA transfection was successful.The proportion of apoptotic cells in the MSC ? Mock group was significantly decreased compared with that in the Ara?C group(12.96 ± 0.95 vs.25.66 ± 1.54%;P<0.001;n=9).Knockdown of FAP α using si RNA weaken the protective effect.The proportion of apoptotic cells in the FAP α?si RNA group was significantly increased compared with the proportion in the NC?si RNA group(22.69 ± 1.99 vs.13.29 ± 1.10%;P<0.001;n=9).The proportion of apoptotic cells in FAP α?si RNA group was also decreased compared with that in the Ara?C group(22.69 ± 1.99 vs.25.66 ± 1.54%;P=0.001).5.The expression of β?catenin in Kasumi?1 cells were significantly increased w hen Kasumi?1 cells were cocultured with BMMSCs and NC?si RNA for 48 h,and even in the presence of Ara?C the expression of β?catenin is activated.Compared with NC?si RNA,knockdown of FAP α using si RNA significantly suppressed the expression of β?catenin.In the absence of BMMSCs,the expression of β?catenin was-decreased from(113.20 ± 2.77)to(44.02 ± 0.06)in groups with and without Ara?C,respectively(P<0.0001).In addition,in the presence of Ara?C and BMMSCs,the expression of β?catenin in the NC?si RNA group and FAP α?si RNA group was(116.84 ± 0.40)and(82.11 ± 1.26),respectively(P<0.0001).6.In the absence of BMMSCs,the addition of β-catenin signaling inhibitor XAV939 had no significant effect on the apoptosis of Kasumi-1 cells.The ratio of apoptosis of Kasumi-1 cells + Ara-C group and Kasumi-1 cells + Ara-C+ XAV939 group was(25.66 ± 1.54%)vs(26.32 ± 1.00%)(P=0.2933),respectively.In the presence of BMMSCs,the addition of β-catenin signaling inhibitor XAV939 significantly increased the ratio of apoptosis of Kasumi-1 cells.The ratio of apoptosis of Kasumi-1 cells + Ara-C + BMMSCs-NC-si RNA group and Kasumi-1 cells + Ara-C + BMMSCs-NC-si RNA + XAV939 group were(13.29 ± 1.10%)vs(15.17 ± 1.01%)(P=0.0017),respectively.The ratio of apoptosis of Kasumi-1 cells + Ara-C + BMMSCs-FAPα-si RNA group and Kasumi-1 cells + Ara-C+ MSCS-FAPα-si RNA + XAV939 group were(22.69 ± 1.99%)vs(24.69 ± 0.95%)(P=0.015),respectively.Conclusion: 1.BMMSCs of AML patients overexpress the FAPα phenotype of CAFs;2.FAPα is highly expressed in AML patients,which may play an important role in AML bone marrow microenvironment.The increased expression of Fapα in BM may be a factor for the poor prognosis of AML;3.BMMSCs in AML patients can protect AML cells from chemotherapy-induced apoptosis,and this protective effect is partly related to the up-regulation of FAPα expression,which is mediated by the β-catenin signaling pathway. |
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