Objective: To investigate the effects of the immunoglobulin G1 heavy chain constant region (IGHG1) on the proliferation and apoptosis of acute myeloid leukemia (AML) THP-1 cells and its possible mechanism of action.
Methods: Human AML THP-1 cells were cultured in vitro and divided into control (normally cultured THP-1 cells), pcDNA3.1 ［THP-1 cells transfected with IGHG1 overexpression (pcDNA3.1-IGHG1) negative control plasmid］, pcDNA3.1-IGHG1 (THP-1 cells transfected with plasmid containing pcDNA3.1-IGHG1), LY364947 ［transforming growth factor-β (TGF-β)/signal transduction protein (Smad) inhibitor LY364947 20 μmol/L treated THP-1 cells］, si-NC ［THP-1 cells transfected with IGHG1-small interfering RNA (siRNA) negative control］, si-IGHG1 (THP-1 cells transfected with IGHG1-siRNA), and si-IGHG1+LY364947 (IGHG1-siRNA and LY364947 co-treated THP-1 cells) a total of 7 groups. Fluorescence quantitative PCR was used to detect the expression of IGHG1 and immunoglobulin G (IgG) mRNA of THP-1 cells in each group; CCK-8 was used to detect THP-1 cells proliferation activity; flow cytometry was used to detect THP-1 cells apoptosis and cell cycle in each group; Western blot was used to detect the THP-1 cells proliferation, apoptosis and the expression of TGF-β/Smad signaling pathway related proteins.
Results: Compared with the control group, after overexpression of IGHG1, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expressions of Cyclin D1, B cell lymphoma-2 (Bcl-2), IgG, TGF-β1, phosphorylated Smad3 (p-Smad3)/Smad3 protein in THP-1 cells were significantly increased (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expression of p21, Bcl-2 related X protein (Bax), Caspase-3 protein were significantly reduced (P<0.05); after inhibiting TGF-β/Smad signaling pathway or silencing IGHG1, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expression of Cyclin D1, Bcl-2, IgG, TGF-β1, p-Smad3/Smad3 protein in THP-1 cells were significantly reduced (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expressions of p21, Bax, and Caspase-3 protein were significantly increased (P<0.05); and compared with silencing IGHG1, after co-treatment of IGHG1 gene silencing and TGF-β/Smad pathway inhibition, the expression of IGHG1 and IgG mRNA, cell proliferation viability, S phase cell ratio, expressions of Cyclin D1, Bcl-2, IgG, TGF-β1, p-Smad3/Smad3 protein in THP-1 cells were significantly reduced (P<0.05), the apoptosis rate, G0/G1 phase cell ratio, expression of p21, Bax, and Caspase-3 protein were significantly increased (P<0.05).
Conclusion: Silencing IGHG1 gene can down-regulate the expression of IgG, inhibit the proliferation of human AML THP-1 cells, block cell cycle progression, and induce cell apoptosis; its mechanism may be related to the inhibition of the TGF-β/Smad pathway activation.
题目: IGHG1对人急性髓系白血病THP-1细胞增殖、凋亡的影响.
目的: 探讨免疫球蛋白G1重链恒定区（IGHG1）对急性髓系白血病（AML）细胞系THP-1细胞增殖、凋亡的影响及其可能的作用机制.
方法: 体外培养人AML THP-1细胞，分为对照（正常培养的THP-1细胞）、pcDNA3.1［转染IGHG1过表达（pcDNA3.1-IGHG1）阴性对照质粒的THP-1细胞］、pcDNA3.1-IGHG1（转染pcDNA3.1-IGHG1的THP-1细胞）、LY364947［转化生长因子-β（TGF-β）/信号转导蛋白（Smad）抑制剂LY364947 20 μmol/L处理THP-1细胞）］、si-NC［转染IGHG1小干扰RNA（IGHG1-siRNA）阴性对照的THP-1细胞］、si-IGHG1（转染IGHG1-siRNA的THP-1细胞）和si-IGHG1+LY364947（IGHG1-siRNA和LY364947共同处理THP-1细胞）共7组。荧光定量PCR法检测各组THP-1细胞中IGHG1和免疫球蛋白G（IgG） mRNA的表达；CCK-8法检测各组THP-1细胞增殖活力；流式细胞术检测各组THP-1细胞凋亡率和细胞周期变化；蛋白印迹法检测各组THP-1细胞增殖、凋亡及TGF-β/Smad信号通路相关蛋白的表达.
结果: 与对照组相比，过表达IGHG1后THP-1细胞中IGHG1和IgG mRNA表达、细胞增殖活力、S期的细胞比例、细胞周期蛋白D1（Cyclin D1）、B细胞淋巴瘤-2（Bcl-2）、IgG、TGF-β1、磷酸化Smad3（p-Smad3）/Smad3蛋白表达均显著升高（P＜0.05），细胞凋亡率、G0/G1期的细胞比例、p21、Bcl-2相关X蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）蛋白表达均显著降低（P＜0.05）。抑制TGF-β/Smad信号通路或沉默IGHG1后THP-1细胞中IGHG1和IgG mRNA表达、细胞增殖活力、S期的细胞比例、Cyclin D1、Bcl-2、IgG、TGF-β1、p-Smad3/Smad3蛋白表达均显著降低（P＜0.05），细胞凋亡率、G0/G1期的细胞比例、p21、Bax、Caspase-3蛋白表达均显著升高（P＜0.05）；且与沉默IGHG1相比，IGHG1基因沉默和TGF-β/Smad通路抑制共同处理的THP-1细胞中IGHG1和IgG mRNA表达、细胞增殖活力、S期的细胞比例、Cyclin D1、Bcl-2、IgG、TGF-β1、p-Smad3/Smad3蛋白表达均显著降低（P＜0.05），细胞凋亡率、G0/G1期的细胞比例、p21、Bax、Caspase-3蛋白表达均显著升高（P＜0.05）.
结论: 沉默IGHG1基因可下调IgG的表达，抑制人AML THP-1细胞增殖，阻滞细胞周期进程，并诱导细胞凋亡；其作用机制可能与抑制TGF-β/Smad通路的激活有关.
Keywords: acute myeloid leukemia THP-1 cells; apoptosis; immunoglobulin G; immunoglobulin G1 heavy chain constant region; proliferation; transforming growth factor-β/signal transduction protein.