| Objective Through researching the relationship between Blimp1 and ATF4/CHOP apoptosis pathway induced by endoplasmic reticulum stress in normal plasma cells and malignant plasma cells, we investigated the anti-apoptosis mechanism of Blimp1 in MM cells. Through stimulating U266 cells by aspirin, we discussed the anti-myeloma mechanism of aspirin.Methods(1) Collect bone marrow fluid of 40 cases of newly diagnosed multiple myeloma patients without treatment and 30 cases of control person with relatively normal bone marrow, whose bone marrow cell morphology showed normal or idiopathic thrombocy reduce purpura or iron deficiency anemia. CD138+ plasma cells were separated and enriched by immunomagnetic beads method and the m RNA expression levels of Blimp1, ATF4 and CHOP in plasma cells were detected by real-time fluorescence quantitative polymerase chain reaction.(2) The U266 myeloma cells were divided into blank control group, negative control group(with negative control vector) and positive control group(with(LV-Blimp1-RNAi(40051-2) lentiviral expression vector). After transfection 72 h, the m RNA expression levels of Blimp1, ATF4 and CHOP of three groups were detected by real-time fluorescence quantitative polymerase chain reaction.(3) U266 cells were stimulated by aspirin solution with different concentrations(0, 0.5, 2.5, 5.0mmol/L) in vitro. Then the effect of aspirin on U266 cells proliferation was measured by CCK-8 assay, and the inhibition rate was calculated.(4) U266 cells were stimulated by aspirin solution with different concentrations(0, 0.5, 2.5, 5.0mmol/L) in vitro. After 48 h, the m RNA expression levels of Blimp1, ATF4 and CHOP of four groups were detected by real-time fluorescence quantitative polymerase chain reaction. SPSS 17.0 and Graph Pad Prism 5 software were used for statistical analysis, and all the information and data were normally distributed with mean ± standard deviation. Two independent samples were compared using independent sample t test, multiple sets of quantitative data were compared using one-way analysis of variables, and between any two groups were compared using Bonferroni. α=0.05 was considered statistically significant.Results 1. Expression levels of Blimp1, ATF4 and CHOP in normal plasma cells and multiple myeloma cells: Compared with normal plasma cells, the Blimp1 m RNA expression level of plasma cells isolated from multiple myeloma patients was significantly increased and the difference was considered statistically significant(P < 0.05), and the m RNA expression levels of ATF4 and CHOP significantly decreased and the difference was statistically significant(P < 0.05). 2. Effect of LV-Blimp1-RNAi on the expression levels of Blimp1, ATF4 and CHOP in U266 cells infected with virus vector 72h: The optimum MOI value of U266 cells is 100. In the case of MOI=100, thetransfection efficiency is beyond 80% under fluorescence microscope. Compared with control groups, Blimp m RNA expression level in positive group significantly reduced and the expression levels of ATF4 and CHOP were significantly increased, and the differences were statistically significant. 3. The effect of different concentration of aspirin on the proliferation of U266 myeloma cells: In the aspirin concentration range of 0.5-5mmol/L, the proliferation activity of U266 myeloma cells was been obviously inhibited with time dependent and concentration dependent. 4. The effect of different concentration of aspirin on the expression of Blimp1, ATF4 and CHOP: After stimulate 48 h with different concentrations aspirin, the expression levels of Blimp1 in U266 cells decreased with the increase of drug concentration, while the expression levels of ATF4 and CHOP increased with the increase of drug concentration.Conclusions 1. Blimp1 can inhibit the apoptosis of myeloma cells induced by endoplasmic reticulum stress by interfering with ATF4/CHOP signaling pathway. 2. By inhibiting the expression of Blimp1 and promoting the expression of ATF4 and CHOP, aspirin can exert anti-myeloma cells. |
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