Objective: To explore the expression of Blimp1, ATF4 and CHOP in bone marrow mononuclear cells from patients with multiple myeloma as well as the effect of aspirin on their expression.
Methods: Sixty untreated patients with multiple myeloma and 30 patients with relatively normal bone marrow were selected. Mononuclear cells from the bone marrow fluid were separated using Ficoll separation solution. CD138+ plasma cells were sorted by immunomagnetic beads method. RT-PCR was used to detect the expression levels of Blimp1, ATF4 and CHOP mRNA in U266 cells cultured in vitro. The cells were divided into blank control group, negative control group (no-loaded virus transfection) and positive experimental group [LV-Blimp1-RNAi (40051-2) transfection] by lentivirus transfection. RT-PCR was used to detect the expression of Blimp1, ATF4 and CHOP mRNA in cells of different groups. U266 cells were stimulated in vitro with different concentrations of aspirin solution (0, 0.5 mmol/L, 2.5 mmol/L, 5.0 mmol/L) for 24, 48 h and 72 h, respectively. The ability of cell proliferation in different groups was measured by CCK-8. U266 cells were stimulated with different concentrations of aspirin for 48 hours. And the mRNA expression of Blimp1, ATF4 and CHOP was detected by RT-PCR.
Results: Compared with plasma cells in normal group, the expression of Blimp1 mRNA in CD138+ plasma cells of MM patients significantly increased (8.040±1.878), and the mRNA expression levels of ATF4 and CHOP significantly decreased (0.735±0.089; 0.837±0.062) (P＜0.05). U266 cells were cultured in vitro. Compared with the blank control group and the negative control group, the mRNA expression level of Blimp in the positive experimental group was significantly down-regulated after infection with LV-Blimp1-RNAi (40051-2) lentiviral expression vector (0.637±0.021). ATF4 and CHOP mRNA expression levels were significantly increased (1.452 ± 0.027; 1.721 ± 0.038) (P＜0.05). The proliferation of U266 cells decreased after stimulation with aspirin. In the range of (0.5-5) mmol/L, aspirin could significantly inhibit the proliferation of U266 cells. The inhibition effect of aspirin was increased along with prolongation of time and increase of concentrations. After aspirin stimulation of different concentrations for 48 hours, the expression level of Blimp1 in U266 cells decreased with increasing of drug concentration, while the expression levels of ATF4 and CHOP increased with increasing of drug concentration.
Conclusion: Inhibition of Blimp1 expression in multiple myeloma cells can promote the expression of ATF4 and CHOP. Aspirin can inhibit the proliferation activity of myeloma cells by down-regulating Blimp1 expression in myeloma cells and up-regulating ATF4 and CHOP expression, therefore plays an anti-tumor rote.
题目: 多发性骨髓瘤细胞中Blimp1、ATF4和CHOP的表达及阿司匹林对其表达的影响.
目的: 探究多发性骨髓瘤（MM）患者骨髓单个核细胞中Blimp1、ATF4因子和CHOP蛋白的表达及阿司匹林对其表达的影响及意义.
方法: 采用Ficoll分离液分离60例初诊未经治疗的多发性骨髓瘤患者和30例骨髓相对正常者骨髓液中的单个核细胞,免疫磁珠法分选CD138+的浆细胞并通过实时荧光定量PCR检测细胞中Blimp1，ATF4和CHOP mRNA表达水平。体外培养U266细胞，使用慢病毒感染细胞，采用实时荧光定量PCR检测不同分组细胞中Blimp1，ATF4和CHOP mRNA表达水平。用不同浓度的阿司匹林溶液（0、0.5、2.0和5.0 mmol/L）体外分别刺激U266细胞24、48和72 h，采用CCK-8法检测阿司匹林对细胞的增殖活性的抑制作用。收集不同浓度的阿司匹林溶液刺激48 h后的U266骨髓细胞，实时荧光定量PCR检测各组细胞中Blimp1、ATF4和CHOP mRNA的表达水平.
结果: 与相对正常组浆细胞比较，MM患者CD138+浆细胞中Blimp1 mRNA表达水平显著升高（8.040±1.878），ATF4和CHOP mRNA表达水平显著降低（0.735±0.089；0.837±0.062）（P＜0.05）。体外培养U266细胞，相较空白对照组与阴性对照组，经LV-Blimp1-RNAi（40051-2）慢病毒表达载体感染后阳性实验组中Blimp mRNA的表达水平显著下调（0.637±0.021），ATF4和CHOP mRNA表达水平显著上调（1.452±0.027；1.721±0.038）（P＜0.05）。CCK-8检测不同浓度阿司匹林刺激24、48和72 h后，U266细胞增殖活性降低，阿司匹林（0.5-5）mmol/L浓度范围内可显著抑制U266细胞的增殖活性，抑制作用随药物刺激时间的延长和刺激浓度的增加而增强（r24 h=0.986; r48 h=0.951; r72 h=0.954; r0.5=0.997; r2.5=0.997; r5.0=0.974）。不同浓度阿司匹林刺激48 h后，U266细胞中Blimp1 mRNA的表达水平随药物浓度升高而依次降低（r=-0.981），而ATF4和CHOP mRNA的表达水平随药物浓度升高而依次升高（rATF4=0.973; rCHOP=0.995）.
结论: 抑制多发性骨髓瘤细胞中Blimp1的表达，可促进ATF4和CHOP的表达。阿司匹林可通过下调骨髓瘤细胞中Blimp1表达和上调ATF4和CHOP的表达抑制骨髓瘤细胞的增殖活性，发挥抗肿瘤活性.