| Multiple myeloma（MM）is a malignant tumor that originates from abnormal proliferation of B lymphocytes.It is characterized by abnormally increased monoclonal immunoglobulin production in the blood and urine.MM is still an incurable hematological malignancy for most patients.The MutT homologue-1 enzyme（MutT homolog 1,MTH1）can hydrolyze 8-oxo-dGTP and 2-OH-dATP and prevent them from participating in DNA replication,and protect tumor cells from damage and aging.Objective:（1）To detect the expression of MTH1（Mut T homolog 1,MTH1）in CD138 negative cells and CD138 positive cells in patients with multiple myeloma（multiple,myeloma,MM）.（2）To explore the effect of MTH1 inhibitor TH588 on the morphology,proliferation and apoptosis of multiple myeloma cell line U266 cells.（3）To explore the effect of IL-6 on the expression level of MTH1 of MM cell line initially.Methods:（1）Obtain and separate bone marrow samples from newly diagnosed patients with multiple myeloma,obtain mononuclear cells,sort and obtain patient CD138negative cells and CD138 positive cells and extract RNA.Using real-time PCR technology to detect the expression of Nudix family members MTH1,NUDT2,NUDT5and NUDT21 of CD138 negative cells and CD138 positive cells in myeloma patients.（2）Observe the effect of IL-6 on the change of MTH1 expression after IL-6 application in multiple myeloma cell line U266.（3）Multiple myeloma U266 cells were treated with different concentrations of MTH1 inhibitor TH588.After 48 hours,the morphological changes of U266 cells were observed under the microscope.DAPI staining was used to observe the morphology changes of the nucleus under the different concentrations of TH588 on multiple myeloma U266 cells after 48 hours.（4）Luciferase activity was used to detect the effect of different concentrations of TH588 on the proliferation of multiple myeloma cell line U266 cells.（5）Flow cytometry was used to detect the effect on apoptosis treating with different concentrations of TH588 on multiple myeloma U266cells after 48h.Results:（1）The expression of MTH1,NUDT2,NUDT5 and NUDT21 in CD138-positive cells of patients with multiple myeloma was higher than that of CD138-negative cells,and all had statistical significance（P<0.05）.（2）According to the analysis of QPCR results,2-ΔCT value of the MTH1 expression of multiple myeloma cell line U266 without IL-6 applied is 0.01775±0.0008,when the concentration of IL-6 is25ng/ml,2-ΔCTΔCT value is 0.04370±0.0016;when the IL-6 concentration is 100ng/ml,2-ΔCT value is 0.02596±0.0008.Compared with the control group,the expression level of MTH1 in U266 cells using IL-6 is increased to varying degrees,and it has statistically significance（P<0.01）.（3）Luciferase reporter gene results showed that there was no statistically significant difference compared with the control group after 48 hours of TH588 on multiple myeloma U266 cell line when the drug concentration was 1μmol/L.When the drug concentration reached 2.5μmol/L,the proliferation inhibition effect was obvious,and the difference was statistically significant（P<0.05）.The higher the drug concentration,the lower the fluorescence value（r=-0.93）.After 72 hours of application,compared with the control group,the drug concentration of 1μmol/L had the effect ofinhibiting cell proliferation（P<0.05）.As the drug concentration further increased,the inhibitory effect was more obvious（r=-0.91）.（4）After 48h treatment of U266 cells with TH588,under the microscope,the cells showed shrinkage,irregular shape and so on.After DAPI staining,observed under a fluorescent microscope,the cells treated with TH588 showed shrinking nuclei,uneven staining,petal-like nuclei,radial nuclei and other apoptotic features.（5）According to flow cytometry analysis,after treatment of U266 cells with TH588,the proportion of double negative cells in the control group was 81.93%.When the drug concentration was 1μmol/L,the proportion of double negative cellsdecreased to 74.60%.When the drug concentration is 2.5μmol/L,the double negative ratio drops to 68.53%.When the drug concentration is 5μmol/L,the double negative ratio drops to 67.33%.When the drug concentration was 10μmol/L,the double negativeratio dropped to 65.80%.After statistical analysis,the difference was statistically significant（P<0.05）.Conclusion:（1）MTH1 is highly expressed in CD138 positive cells of some patients with multiple myeloma.（2）MTH1 inhibitor TH588 has a significant effect on inhibiting proliferation and inducing apoptosis in multiple myeloma U266 cell line.（3）IL-6 can increase the expression level of MTH1 in multiple myeloma U266 cells. |
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