Study On The Mechanisms Of Iron Overload Damaging The Bone Marrow Mesenchymal Stem Cell In Patients With Intermediate Or High Risk Myelodysplastic Syndrome And Promoting Leukemia Transformation

Objective: 1.To study the effects of iron overload(IO)on the quantity and functions of bone marrow mesenchymal stem cell(BM-MSC)and the mechanisms of BM-MSC damaged by IO,finally to elucidate the role of IO in prompting leukemia transformation.2.To study the synergistic effects of the combination of arsenic trioxide(ATO)and decitabine(DAC)against MDS cells on the cell viability and apoptosis,in order to investigate the possible synergistic mechanisms in the apoptosis of MDS cells,providing evidences for the use of the combination in MDS patients.Contents: Forty intermediate or high risk MDS patients(eighteen cases in IO MDS group: ferritin≥1000ng/ml,twenty-two cases in NIO MDS group: ferritin<1000 ng/ml),ten IO MDS/AML patients and nineteen normal controls were involved in our study.BM-MSC was induced in vitro,the effects of IO on the quantity and functions of BM-MSC were examined.Meanwhile,the effects of IO on the levels of reactive oxygen species(ROS),apoptosis and related signaling pathways in the BM-MSC were examined,as well as the effects of the antioxidant or iron chelator on the BM-MSC damaged by IO.The mechanisms of IO promoting leukemia transformation were further investigated.MDS MDS cell lines were cultured in vitro,the effects of the combination of ATO and DAC on the proliferation and apoptosis of MDS cell lines were examined,in order to explore the role of endoplasmic reticulum stress related signaling pathway in the synergistic anti-MDS cells of the combination.Methods: Part 1.Study on the mechanisms of iron overload damaging the bone marrow mesenchymal stem cell in patients with intermediate or high risk Myelodysplastic syndrome and promoting leukemia transformation.BMMNC was separated from sterile bone marrow,and BM-MSC was induced in vitro.The morphology and quantity of BM-MSC were observed,and surface markers were detected by flow cytometry(FCM).The proliferation of BM-MSC were examined by CCK-8 kit.Osteogenic differentiation of BM-MSC was induced and identified by silver nitrate staining.The gene expressions regulating hematopoiesis in BM-MSC were examined by RT-PCR,and the expressions of TGF-β1 in the culture supernatants secreted by BM-MSC were detected by Elisa.The levels of ROS and apoptosis of BM-MSC were examined by FCM.The expressions of AKT,apoptosis and Wnt/β-catenin signaling pathways in the BM-MSC were examined by RT-PCR and Western blot.The effects of antioxidant or iron chelator on IO MDS BM-MSC were examined.The differences in gene mutations between IO MDS group and NIO MDS group were compared.Meanwhile,the differences on the levels of ROS,apoptosis and the gene expressions and protein levels above in BM-MSC between IO MDS group and IO MDS/AML group were further compared.Part 2.Study on the mechanism of synergistic effects against MDS cells induced by the combination of ATO and DAC.MUTZ-1 and SKM-1 cells were treated with ATO,DAC and the combination for 72 h,the IC50 values of ATO and DAC in MDS cells were determined respectively,the combination index(CI)of the combination was calculated.The cell apoptosis was examined by FCM.RNA-sequence was performed on the MUTZ-1 cells after the treatment with ATO,DAC or the combination,in order to detect the differentially expressed genes between them.The endoplasmic reticulum(ER)stress related genes and proteins in MDS cells treated with ATO,DAC and the combination were examined by RT-PCR and Western Blot.The levels of ROS in MDS cells were examined by FCM.The levels of ROS and apoptosis in the group of the combination treated with antioxidant in MDS cells were examined by FCM.The ER stress related genes in the group of the combination treated with antioxidant in MDS cells were examined by RT-PCR.Results:Part 1.Study on the mechanisms of iron overload damaging the bone marrow mesenchymal stem cell in patients with intermediate or high risk Myelodysplastic syndrome and promoting leukemia transformation.1.The quantity of BM-MSC in IO MDS group was less than that in NIO MDS group,but there was no significant difference in morphology between them.The purity of BM-MSC was over 90%.The iron deposition of BM-MSC increased in IO MDS group,with the decreased ability of osteogenic differentiation oriented from BM-MSC.The proliferation ability of BM-MSC from IO MDS group and NIO MDS group significantly decreased,and the proliferation ability of BM-MSC significantly decreased in IO MDS group compared with that in NIO MDS group on days 6,8 and 10(p<0.05).The gene expression of VEGFA in IO MDS BM-MSC(0.39±0.46)was significantly lower than that in NIO MDS group(0.81±0.66)and control group(1.21±0.69)(p<0.05).The gene expression of CXCL12 in IO MDS group(0.59±0.66)was significantly lower than that in NIO MDS group(1.11±0.83)and control group(1.20±0.88)(p<0.05).The gene expression of TGF-β1 in IO MDS BM-MSC(0.52±0.45)was significantly lower than that in NIO MDS group(1.02±0.71)and control group(1.16±0.73)(p<0.05).The expression of TGF-β1 in the culture supernatants secreted by BM-MSC in IO MDS group(1871.79±601.87)pg/ml was significantly lower than that in NIO MDS group(2610.59±464.51)pg/ml and control group(3323.03±724.18)pg/ml(p<0.05).2.The level of ROS in IO MDS BM-MSC(1188503.71±151641.12)was significantly higher than that of NIO MDS group(1031773.06±125481.41)and control group(911600.09±134124.83)(p<0.05).The total apoptosis of IO MDS BM-MSC(47.49±11.38)% was significantly higher than that in NIO MDS group(38.16±6.86)% and control group(30.24±8.51)%(p<0.01).The gene expression of caspase3 in IO MDS BM-MSC(1.99±1.07)was significantly higher than that of NIO MDS group(1.44±0.74)and control group(1.13±0.59)(p<0.05).The gene expression of β-catenin was significantly highly expressed in IO MDS BM-MSC(4.64±2.63)than that of NIO MDS group(2.46±1.24)and control group(1.25 ±0.70)(p<0.001).The protein levels of p-AKT,GSK-3β、p-GSK3β in IO MDS BM-MSC were lower than those in NIO MDS group and control group,while the protein levels of caspase3 and β-catenin were higher than those in NIO MDS group and control group.After the treatments with NAC or DFO for 24 h,the levels of ROS,apoptosis,gene expressions and the protein levels of caspase3 and β-catenin in IO MDS BM-MSC significantly decreased,while the protein levels of p-AKT、GSK-3β、p-GSK3β increased.Meanwhile,the gene expressions of VEGFA,CXCL12 and TGF-β1 in IO MDS BM-MSC significantly highly expressed after the treatments of NAC or DFO(p<0.05).3.The mutation frequency of two or more genes in IO MDS group was significantly higher than that in NIO MDS group,and TET2 and ASXL1 gene mutations in IO MDS group were significantly higher than that in NIO MDS group(p<0.05).The quantity of BM-MSC in patients with IO MDS/AML was lower than that in patients with IO MDS,and the protein level of p-AKT significantly decreased.The level of ROS in IO MDS/AML group was significantly higher than that of IO MDS group(p<0.05).But there were no significant differences in apoptosis,gene expressions of VEGFA,CXCL12,and the gene expressions and protein levels of caspase3 and β-catenin in BM-MSC between IO MDS/AML group and IO MDS group(p>0.05).Part 2.Study on the mechanism of synergistic effects against MDS cells induced by the combination of ATO and DAC.1.The IC50 values of ATO and DAC in MUTZ-1 cells for 72 h were 0.699(0.478-1.022)μM and 4.182(3.689-4.740)μM,CI <1.The IC50 values of ATO and DAC in SKM-1 cells for 72 h were 1.457(0.939-2.259)μM and 1.162(0.461-1.422)μM,CI<1.The apoptosis of the combination in MUTZ-1 cells for 72h(57.75±9.87)% was significantly higher than that of the ATO group(27.04±3.85)%,the DAC group(31.89±6.00)% and the control group(7.18±1.39)%(p<0.01).The apoptosis of the combination in SKM-1 cells for 72h(78.57±1.37)% was significantly higher than that of the ATO group(49.91±4.12)%,the DAC group(60.99±2.25)% and the control group(11.64 ±2.47)%(p<0.001).2.RNA-sequence was performed on MUTZ-1 cell line after treatment with ATO,DAC or the combination,in order to detect the differentially expressed genes between them.Combined with GSEA gene function analysis,ER stress-related signaling pathway played an important role in the apoptosis of MUTZ-1 cells induced by the combination.The gene expressions of DDIT3,GRP78,GADD34 and caspase3 in MUTZ-1 cells treated with the combination were significantly higher than those in the control group,the ATO group and the DAC group(p<0.05).The gene expressions of DDIT3,ATF6,GRP78 and caspase3 in SKM-1 cells treated with the combination were significantly higher than those in the control group,the ATO group and the DAC group(p<0.05).The MUTZ-1 cells treated with ATO and DAC significantly increased the levels of ROS(140620.33±8265.45)than that in ATO group(123138.67±5692.58),the DAC group(91830.33±4857.53)and the control group(105255.33±3577.11)(p<0.01).The levels of ROS in SKM-1 treated with the ATO and DAC(204463.00±6750.81)were significantly higher than those in the ATO group(169393.33±14367.41),the DAC group(147279.33±25314.92)and the control group(82094.67±11567.04)(p<0.05).The protein levels of AKT and BCL2 in MDS cells treated with the combination of ATO and DAC were lower than those in the single agent groups and the control group,while the protein levels of CHOP,BIP,ATF6 and caspase3 were higher than those in the single agent groups and the control group.3.The levels of ROS in the group of the combination treated with antioxidant agent in MUTZ-1 cells(118830.00±8429.79)were significantly lower than that in the combination group(140620.33±8265.45)(p<0.01).The levels of ROS in the group of the combination treated with antioxidant agent in SKM-1 cells(141005.33±8671.97)was significantly lower than that in the combination group(204463.00±6750.81)(p<0.001).The apoptosis of MUTZ-1 cells in the combination with NAC group(42.42±7.18)% was significantly lower than that in the combination group(57.75±9.87)%(p<0.05).The apoptosis of SKM-1 cells in the combination with NAC group(67.90±2.31)% was significantly lower than that in the combination group(78.57±1.37)%(p<0.01).The antioxidant treatment significantly reduced the gene expressions of DDIT3,GRP78 and GADD34 in MUTZ-1 cells induced by the combination(p<0.05).The antioxidant treatment significantly reduced the gene expressions of ATF6,GRP78 and GADD34 in SKM-1 cells induced by the combination(p<0.05).Conclusions:1.Iron overload significantly affected the quantity and proliferation and osteogenesis differentiation abilities of BM-MSC isolated from intermediate or high risk MDS patients,and inhibited the hematopoietic related gene expressions of VEGFA,CXCL12 and TGF-β1 in BM-MSC.Meanwhile,IO obviously increased the levels of ROS and apoptosis in BM-MSC of intermediate or high risk MDS patents,activated caspase3 and β-catenin related signaling pathways,which could be partially reversed by antioxidant or iron chelator.The frequency of the gene mutations in IO MDS group was higher than that in NIO MDS group.At the same time,IO could further downregulate the protein level of p-AKT in BM-MSC and reduce its quantity in MDS/AML patients.However,IO did not significantly increase the apoptosis of BM-MSC in MDS/AML patients,as well as the expressions of VEGFA,CXCL12,caspase3 and β-catenin.It means that IO affects the quantity and functions of BM-MSC in intermediate or high risk MDS patients,and damages BM-MSC by activating apoptosis and Wnt/β-catenin signaling pathways.IO may affect the genetic stability and damage BM-MSC from intermediate or high risk MDS patients,which may involve in promoting leukemia transformation.2.The combination of ATO and DAC shows synergistic effects against MDS cells by inhibiting cell proliferation and inducing apoptosis of MDS cells.ER-stress related pathways played an important role in the apoptosis of MUTZ-1 induced by the combination through the RNA-sequence.The ER-stress related gene expressions and protein levels in MDS cells were significantly upregulated.Meanwhile,the levels of ROS in MDS cells significantly increased in the combination group.The levels of ROS,apoptosis and the ER-stress related gene expressions in the group of the combination treated with antioxidant in MDS cells were significantly decreased.It means that ATO combined with DAC shows synergistic effects against MDS cells through ER stress-related apoptosis.