Lin Ying, Zhang Rongdong, Jiang Shenghua, Lu Wei, Lin Zenghua, Liu Hong
Department of Hematology, Affiliated Hospital of Nantong University, Nantong University, Nantong, China.
Department of Hematology, Ningde Hospital affiliated to Medical University of Fujian, Ningde, China.
Transl Cancer Res. 2021 Jan;10(1):327-336. doi: 10.21037/tcr-20-1866.
Interferon regulatory factor-1 (IRF-1) plays a critical role in the injury to stem and progenitor regions associated with aberrant interferon-gamma (IFN-γ) in aplastic anemia (AA). The present study aimed to investigate the effects of IFN-γ on murine myeloid precursor cells (32D cells) with wild-type and inactive-type protein kinase B (Akt) after gene silencing.
With treatment of four concentrations of IFN-γ, the 32D cell viability and inhibition rate were assayed by middle-time-spray (MTS). The apoptosis rate was determined by flow cytometry, and the expression of the phosphorylated signal transducer and activator of transcription 3 (p-Stat3) and the phosphorylated signal transducer and activator of transcription 5 (p-Stat5) was analyzed by Western blot.
The results from real time PCR (RT-PCR) assays suggested that the relative expression level of IRF-1-mRNA in the knockdown group (KD) was lower than that of in the negative control (NC) and blank control (Ctrl). In addition, the silencing efficiency was >70%, which was further validated by Western blotting. At 48 h, the rate of proliferation of 32D cells of wild-type Akt was significantly higher than that of inactive-type Akt (0.918±0.005 0.503±0.003, P=0.008), while the apoptosis rate in wild-type was significantly lower than that of inactive Akt (1.46%±0.41% 2.98%±0.32%, P=0.006). After reducing the expression of gene, the promotion of hematopoiesis was recovered, resulting from the high concentration of IFN-γ achieved by reducing the expression of p-Stat5 via the Akt signaling pathway.
Taken together, these results suggested that IRF-1 plays a critical role in the pro-apoptotic effect of IFN-γ on the proliferation of hematopoietic progenitor cells. These findings could contribute to understanding the mechanisms underlying the conversion from IFN-γ-mediated inhibition to promotion of hematopoiesis.
干扰素调节因子-1（IRF-1）在再生障碍性贫血（AA）中与异常干扰素-γ（IFN-γ）相关的干细胞和祖细胞区域损伤中起关键作用。本研究旨在探讨基因沉默后IFN-γ对野生型和无活性型蛋白激酶B（Akt）的小鼠骨髓前体细胞（32D细胞）的影响。
用四种浓度的IFN-γ处理后，通过中时喷雾（MTS）法检测32D细胞活力和抑制率。通过流式细胞术测定凋亡率，并通过蛋白质印迹法分析磷酸化信号转导和转录激活因子3（p-Stat3）和磷酸化信号转导和转录激活因子5（p-Stat5）的表达。
实时荧光定量聚合酶链反应（RT-PCR）检测结果表明，敲低组（KD）中IRF-1-mRNA的相对表达水平低于阴性对照组（NC）和空白对照组（Ctrl）。此外，沉默效率>70%，蛋白质印迹法进一步验证了这一点。48小时时，野生型Akt的32D细胞增殖率显著高于无活性型Akt（0.918±0.005对0.503±0.003，P=0.008），而野生型的凋亡率显著低于无活性Akt（1.46%±0.41%对2.98%±0.32%，P=0.006）。基因表达降低后，造血促进作用得以恢复，这是通过Akt信号通路降低p-Stat5表达从而实现高浓度IFN-γ所致。
综上所述，这些结果表明IRF-1在IFN-γ对造血祖细胞增殖的促凋亡作用中起关键作用。这些发现有助于理解IFN-γ介导的造血抑制向促进转化的潜在机制。
