| ObjectiveTo explore the molecular markers of the abnormal hematopoietic clone in myelodysplastic syndromes and its related mechanisms involving immunophenotyping and cytogenetics.MethodsSixty-three cases of MDS, thirteen cases of acute myeloid leukemias as well as forty normal controls were enrolled in this study. First Section The expressions of CD96and CD123on CD34+CD38-bone marrow cells(BMC) of these patients and controls were measured by FACS.CD114(GCSFR), EPOR and CD110(TPOR) expression on their CD34+CD38-CD96+and CD34+CD38"CD96" BMC and these cells’ apoptosis(Annexin V) were also detected by FACS. Second Section The expression of TET2mRNA in the bone marrow cells of cases with MDS and normal controls were measured by RT-PCR, aiming to search for the cytogenetic marker of malignant clone in MDS. CD3+T cells and CD34+cells were sorted by magnetic activated cell-sorting system. The mRNA expressions of TET2and DLK1in bone marrow CD3+T cells and CD34+cells were detected by qPCR. Third Section Normal CD34+cells were sorted by magnetic activated cell-sorting system. The silencing effect in vitro of RNA interference (RNAi) on TET2expression in normal CD34+cells was identified by qPCR and Western blot analysis. The cell cycle kinetics and apoptosis of these cells were detected by flowcytometry. Fourth Section CD34+CD38-cells in MDS were sorted by magnetic activated cell-sorting system. The silencing effect in vitro of RNA interference (RNAi) on DLK1expression in CD34+CD38-cells of MDS patients was identified by qPCR and Western blot analysis. The cell cycle kinetics and apoptosis of these cells were detected by flowcytometry.ResultsFirst Section MDS patients had significant higher proportion of CD34+CD38-/CD34+BMC((22.72±23.91)%) than normal controls ((8.63±9.35)%)(P<0.01). The expressions of CD34+CD38-CD96+/CD34+CD38-BMC and CD34+CD38-CD123+/CD34+CD38-BMC of MDS patients ((21.18±24.06,31.62±28.42)%) were significantly higher than those of normal controls ((11.23±17.91,9.29±12.63)%)(P<0.01). The expressions of CD34+CD38-CD96+/CD34+CD38-BMC and CD34+CD38-CD123+/CD34+CD38BMC in high-risk MDS group((26.25±27.64,37.56±31.01%) was significantly higher than those in low-risk group((11.44±10.88,15.53±10.96%)(P<0.05). The expression levels of CD123and CD96were significantly correlated with the proportion of bone marrow blasts(r=0.384, P<0.05; r=0.625, P<0.05).The expressions of CD114, CD110and Annexin V in CD34+CD38-CD96+BMC ((7.23±10.25,0.52±1.23,2.96±3.84)%) were significantly lower than those in CD34+CD38-CD96-BMC ((24.35±21.74,12.03±10.91,13.69±17.98)%) of MDS patients (P<0.05).Second Section The expression of TET2mRNA was down-regulated in MDS compared with controls(P<0.05). There was no significant difference in expression of TET2among RA, RCMD and RAEB cases. Patients with higher expression of TET2(≥0.9) presented significantly lower proportion of bone marrow blasts (1.04±1.68)%than those with lower expression (<0.9) of TET2(6.13±8.17)%(P<0.05). The expression of TET2mRNA in BMMNC of MDS patients was significantly positively correlated with their malignant clone burden (r=-0.398, P<0.05) and IPSS (r=-0.480, P<0.05). The expression of TET2mRNA in CD3+T cells was down-regulated in MDS (0.16±0.15) fold compared with that in normal controls (P<0.05). The expression of TET2mRNA in CD3+T cells of MDS patients was positively correlated with serum complement C3(r=0.404, P<0.05). The expression of DLK1mRNA in CD3+T cells was up-regulated in MDS (1.61±0.88) fold compared with that in controls (P<0.05). Grouped by karyotype, patients with abnormal karyotype presented significantly (1.45±0.44) fold higher of DLK1mRNA expression than those with normal karyotype (P<0.05). The expression of DLK1mRNA in CD3+T cells of MDS patients was positively correlated with their proportion of bone marrow blasts (r=0.343, P<0.05). The expression of TET2mRNA in CD34+cells was down-regulated in MDS compared with that in normal controls [(0.58±0.26)vs(1.25±0.94)](P<0.005). The expression of DLK1mRNA in CD34+cells was up-regulated in MDS compared with that in normal controls[(2.72±1.02)vs(1.31±1.00)](P<0.001).Third Section The siRNA targeting TET2suppressed the expression of TET2in normal CD34+cells in vitro. Meanwhile, the proliferation activity was significantly enhanced [G0/G1phase:(90.82±8.25)%,(92.65±7.06)%,(93.60±5.54)%; S phase:(8.50±8.31)%,(6.92±7.04)%,(5.95±5.53)%; G2/M phase:(0.47±0.89)%,(0.30±0.42)%,(0.53±1.07)%. G0/G1phase and S phase was significantly different between control cells and cells which transfected with TET2siRNA (P<0.005)] And the apoptosis rate was declined in cells which transfected with TET2siRNA as compared to those of the cells transfected with scrambled siRNA and control cells [(23.28±9.73)%vs (26.20±9.78)%vs (26.17±9.88)%, the apoptosis rate was statistically significant between control cells and cells which transfected with TET2siRNA (P<0.05)].Fourth Section The siRNA targeting DLK1suppressed the expression of DLK1in MDS patients’CD34+CD38" cells. Meanwhile, the proliferation activity was significantly reduced [G0/G1phase:(95.81±3.87)%,(91.29±10.39)%,(91.22±10.82)%; S phase:(3.90±3.61)%,(8.41±10.33)%,(8.38±10.65)%; G2/M phase:(0.29±0.46)%,(0.34±0.49)%,(0.40±0.55)%. G0/G1phase and S phase was statistically significant between control cells and cells which transfected with DLK1siRNA (P<0.05)]. And the apoptosis rate was inclined in cells which transfected with DLK1siRNA as compared to those of the cells transfected with scrambled siRNA and control cells [(38.97±14.32)%vs(32.94±12.64)%vs (33.48±12.44)%], the apoptosis rate was statistically significant between control cells and cells which transfected with DLK1siRNA (P<0.05)].Conclusions(1) There were higher proportion of CD34+CD38-CD96+and CD34+CD38-CD123+cells in bone marrow of MDS patients. Those cells expressed lower cytokine receptors and displayed lower apoptosis. Leukemia stem cells(LSC) in MDS might be contained in those cells.(2) The mRNA expressions of TET2in BMMNC、CD3+T cells and CD34+cells of MDS patients decreased, and the mRNA expression of DLK1in CD3+T cells and CD34+ cells was increased, which might useful as an important indicator for the evaluation of MDS clone burden and an important indicator to explore the mechanism of immune tolerance.(3) Suppression of TET2expression renders the CD34+cells harboring more aggressive phenotype. This preliminary finding suggests that CD34+cells lowering expression of TET2may play an oncogenic role on myeloid tumor.(4) Suppression of DLK1expression renders the CD34+CD38" cells harboring lower aggressive phenotype. This preliminary finding suggests that CD34+CD38-cells with over-load expression of DLK1may play an oncogenic role in myeloid tumor. |
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