| Objective:To explore the relationship between multiple myeloma(MM)and histone methylase G9a,and provide a new theoretical basis and thought for the treatment of MM.Methods:A total of 32 newly diagnosed MM patients in the Department of Hematology,General Hospital of Tianjin Medical University from July 2018 to November 2018and 12 normol control(IDA patients)were enrolled.Of these.CD138~+cells from normal control and MM patients were isolated by MACS.The RT-PCR technique was used to analyze the differences in the relative expression of G9a mRNA in CD138~+from patients or myeloma cell lines.Myeloma cell lines(OPM-2 and U266)were treated with G9a specific inhibitor BIX-01294 with different concentrations(0?M?0.625?M,1.25?M,2.5?M,5?M,10?M)at different time(24h,48h).Apoptosis and proliferation of Myeloma cell lines(OPM-2 and U266)were detected by CCK-8 and flow cytometry.Western blot(WB)was used to detect the expression level of G9a and H3K9me2 protiens.Results:Part 1 The mRNA level of G9a in MM patiensThe relative mRNA level of G9a in patients with MM was significantly higher than that in the control group(Median 2.582 vs.0.965,p<0.05),and the G9a level in Myeloma cell lines(OPM-2 and U266)was higher than that of the control(Median4.180 vs.0.965 and 3.965 vs.0.965,p<0.05).However,there was no significant difference between MM patients and Myeloma cell lines.Part 2 Cell proliferation detected by CCK-8BIX-01294 was applied to OPM-2 cell group at concentrations of 0.625?M,1.25?M,2.5?M,5?M and 10?M for 24 hours,and cell viability rates were(96.15±2.864%),(94.17±3.082%),(73.90±1.236%),(49.91±3.117%)and(29.15±1.257%).There was no significant difference in the viability rates of cell proliferation between the experimental group and the control group at low concentrations(0.625?M,1.25?M,p>0.05).Statistically significant differences were observed between experimental group with high concentrations(2.5?M,5?M and 10?M)and the control group(p<0.05).The cell viability rates of cell proliferation at 48h were(96.26±2.626%),(94.94±1.736%),(58.68±2.032%),(25.88±2.068%)and(8.316±0.5757%).There was no significant difference in the viability rates of cell proliferation between the experimental group and the control group at low concentrations(0.625?M,1.25?M,p>0.05).Statistically significant differences were found between experimental group with high concentrations(2.5?M,5?M and10?M)and the control group(p<0.05).The cell viability rates of 24 h and 48 h groups were compared between2.5?M group(73.90±1.236%vs.58.68±2.032%),5?M group(49.91±3.117%vs.25.88±2.068%)and 10?M group(29.15±1.257%vs.8.316±0.5757%),p<0.05,The viability rates increases with the prolongation of the time of action.BIX-01294 was applied to U266 cell group at concentrations of 0.625?M,1.25?M,2.5?M,5?M and 10?M for 24 hours,and cell viability rates were(88.51±8.085%),(74.40±3.457%),(47.00±1.024%),(30.34±1.379%)and(16.78±1.357%).There was no significant difference in the viability rates of cell proliferation between the experimental group and the control group at low concentrations(0.625?M,p>0.05).Statistically significant differences were observed between experimental group with high concentrations(1.25?M,2.5?M,5?M and 10?M)and the control group(p<0.05).The cell viability rates of cell proliferation at 48h were(89.08±6.911%),(78.53±4.249%),(26.08±1.566%),(20.71±3.154%)and(8.856±3.230%).There was no significant difference in the viability rates of cell proliferation between the experimental group and the control group at low concentrations(0.625?M,p>0.05).Statistically significant differences were found between experimental group with high concentrations(1.25?M,2.5?M,5?M and 10?M)and the control group(p<0.05).The cell viability rates of 24 h and48 h groups were compared between 2.5?M group(47.00±1.024%vs.26.08±1.566%),5?M group(30.34±1.379%vs.20.71±3.154%)and 10?M group(16.78±1.357%vs.8.856±3.230%),p<0.05,The viability rates increases with the prolongation of the time of action.Part 3 Cell apoptosis detected by flow cytometryBIX-01294 was applied to OPM-2 cell group at concentrations of 0?M,0.625?M,1.25?M,2.5?M,5?M and 10?M,and the apoptosis rate was(6.507±0.2111%),(6.873±0.4281%),(9.967±0.5487%),(42.87±1.532%),(77.64±0.9978%)and(85.83±0.7076%)after 24 hours of treatment.There was no significant difference in the rates of cell apoptosis between the experimental group and the control group at low concentrations(0.625?M,p>0.05).Statistically significant differences were seen between experimental group with high concentrations(1.25?M,2.5?M,5?M and 10?M)and the control group(p<0.05).After 48 hours,the percentage of cell apoptosis was(5.613±0.5733%),(5.697±0.1641%),(11.00±0.5774%),(48.05±2.488%),(85.46±1.082%),and(90.57±0.3012%),BIX-01294(1.25?M,2.5?M,5?M,10?M)group was statistically different from the control group(p<0.05).It is suggested that apoptosis increases with the increase of drug concentration of BIX-01294.The apoptotic rates of 24 h and 48 h groups were compared between 5?M group(77.64±0.9978%vs.85.46±1.082%)and 10?M group(85.83±0.7076%vs.90.57±0.3012%),p<0.05,The apoptotic rate increased with the prolongation of the time.BIX-01294 was applied to U266 cell group at concentrations of 0?M,0.625?M,1.25?M,2.5?M,5?M and 10?M,and the apoptosis rate was(13.47±0.9400%),(17.11±0.6350%),(26.62±1.835%),(47.57±6.305%),(71.81±1.500%)and(84.43±2.375%)after 24 hours of treatment.There was no significant difference in the rates of cell apoptosis between the experimental group and the control group at low concentrations(0.625?M,p>0.05).Statistically significant differences were seen between experimental group with high concentrations(1.25?M,2.5?M,5?M and 10?M)and the control group(p<0.05).After 48 hours,the percentage of cell apoptosis was(15.39±0.7789%),(17.92±0.9357%),(23.55±0.8278%),(76.21±2.058%),(96.06±0.3810%)and(98.12±0.1563%),BIX-01294(1.25?M,2.5?M,5?M,10?M)group was statistically different from the control group(p<0.05).It is suggested that apoptosis increases with the increase of drug concentration of BIX-01294.The apoptotic rates of 24 h and 48 h groups were compared between 2.5?M(47.57±6.305%vs.76.21±2.058%)group,5?M group(71.81±1.500%vs.96.06±0.3810%)and 10?M group(84.43±2.375%vs.98.12±0.1563%),p<0.05,The apoptotic rate increases with the prolongation of the time.Part 4 The protein expression of G9a and H3K9me2 at 48h after treatment with BIX-01294 on OPM-2 cells.According to the results of proliferation and apoptosis,OPM-2 cells were treated with 0?M/2.5?M BIX-01294 for 48 h.After 48h of BIX-01294 on OPM-2 cells,the expression of G9a and H3K9me2 increased gradually than those in the control group(0?M).Conclusions:1.The relative mRNA level of G9a in patients with multiple myeloma was significantly higher than that in normal controls,and the relative mRNA level of G9a in myeloma cell line(OPM-2,U266)was higher than that in control group,but there was no significant difference between MM patients and myeloma cell lines.2.After high concentration of BIX-01294,the cell proliferation was inhibited and the apoptotic ability was gradually enhanced.The inhibition rate of cell proliferation and the rate of apoptosis are concentration-dependent and time-dependent.3.The expression of G9a and H3K9me2 protein in OPM-2 was significantly decreased by BIX-01294 treatment at 2.5?M for 48 h.4.G9a was highly expressed in MM.BIX-01294 could inhibit the proliferation of MM cells and promote apoptosis by significantly reducing the level of H3K9 double methylation.G9a may play a role in the occurrence and development of MM,suggesting that G9a may be a potential therapeutic target for MM. |
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