Zhu Yu-Qian, Wu Ling-Yun
Department of Hematology, The Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China.
Department of Hematology, The Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China,E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2021 Dec;29(6):1858-1863. doi: 10.19746/j.cnki.issn.1009-2137.2021.06.028.
To investigate the effect of U2AF1 gene mutation to inflammatory cytokine in SKM-1 cell of human myelodysplastic syndromes (MDS), and whether the above effects were mediated by FOXO3a-Bim signaling pathway.
Wide-type U2AF1 and mutant U2AF1 (the serine residue 34 was replaced by phenylalanine, and named as S34F) recombinant expression plasmids were constructed. Lentiviruses were packaged and transfected into SKM-1 cells. The expression of FOXO3a was up-regulated by lentiviruses, and its transfection rate was investigated. The cell proliferation was detected by CCK-8 method. Flow cytometry was used to detect the apoptosis and cycle of the cells. The expression pro-inflammatory cytokine IL-1β, IL-6, TNF-α and anti-inflammatory cytokine IL-4 were detected by qRT-PCR. FOXO3a, Bim, Bcl-2 and Bax protein expression levels were detected by Western blot.
Compared with the control group, the cell apoptosis rate, pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly increased in the S34F group (P<0.05); cell cycle was blocked at the G phase; cell proliferation and the anti-inflammatory cytokine IL-4 transcription level were significantly decreased; the expression levels of FOXO3a, Bim and Bax protein were significantly increased (P<0.05); while the expression level of Bcl-2 protein was significantly decreased (P<0.05). The up-regulation of FOXO3a could significantly inhibited the proliferation and increased cell apoptosis of SKM-1 cells with U2AF1 S34F mutation; cell cycle was blocked at the S and G phases; the pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly decreased (P<0.05), and the transcription level of anti-inflammatory cytokine IL-4 showed no statistically significant as compared with control group (P>0.05).
U2AF1 S34F mutation can regulate inflammatory phenotype in SKM-1 cells, which may be mediated through FOXO3a-Bim signaling pathway.
探讨U2AF1基因突变对人骨髓增生异常综合征（MDS）SKM-1细胞中炎性细胞因子的影响，以及上述作用是否由FOXO3a-Bim信号通路介导。
构建野生型U2AF1和突变型U2AF1（丝氨酸残基34被苯丙氨酸取代，命名为S34F）重组表达质粒。包装慢病毒并转染至SKM-1细胞。通过慢病毒上调FOXO3a的表达，并检测其转染率。采用CCK-8法检测细胞增殖。流式细胞术检测细胞凋亡和周期。通过qRT-PCR检测促炎细胞因子IL-1β、IL-6、TNF-α和抗炎细胞因子IL-4的表达。采用蛋白质免疫印迹法检测FOXO3a、Bim、Bcl-2和Bax蛋白表达水平。
与对照组相比，S34F组细胞凋亡率、促炎细胞因子IL-1β和TNF-α转录水平显著升高（P<0.05）；细胞周期阻滞于G期；细胞增殖和抗炎细胞因子IL-4转录水平显著降低；FOXO3a、Bim和Bax蛋白表达水平显著升高（P<0.05）；而Bcl-2蛋白表达水平显著降低（P<0.05）。上调FOXO3a可显著抑制U2AF1 S34F突变的SKM-1细胞增殖并增加细胞凋亡；细胞周期阻滞于S期和G期；促炎细胞因子IL-1β和TNF-α转录水平显著降低（P<0.05），抗炎细胞因子IL-4转录水平与对照组相比无统计学意义（P>0.05）。
U2AF1 S34F突变可调节SKM-1细胞的炎性表型，可能通过FOXO3a-Bim信号通路介导。
