[Effect of U2AF1 Mutation to Inflammatory Cytokine Expression in SKM-1 Cells through FOXO3a-Bim Signaling Pathway].

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 G2 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 G2 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.U2AF1基因突变调控FOXO3a-Bim信号通路对SKM-1细胞炎症因子表达的影响.探讨U2AF1基因突变对人骨髓增生异常综合征（MDS）细胞系SKM-1细胞炎症因子表达的影响，并观察上述作用是否通过FOXO3a-Bim信号通路介导.构建U2AF1野生型以及U2AF1蛋白序列第34位丝氨酸突变为苯丙氨酸的真核表达质粒（S34F），慢病毒包装并转染SKM-1细胞，通过慢病毒技术上调细胞中FOXO3a的表达并验证其转染效率。采用CCK-8法检测各组细胞的增殖；流式细胞术检测细胞周期和细胞凋亡；qRT-PCR法检测促炎因子IL-1β、IL-6和TNF-α,以及抑炎因子IL-4的表达；Western blot法检测FOXO3a、Bim、Bcl-2和Bax蛋白的表达水平.与对照组相比，S34F组细胞凋亡率及促炎因子IL-1β和TNF-α转录水平显著增加（P＜0.05），细胞周期阻滞在G2期，细胞增殖及抑炎因子IL-4的转录水平显著降低；FOXO3a、Bim和Bax蛋白表达水平显著增加（P＜0.05）；Bcl-2表达水平显著降低（P＜0.05）。FOXO3a基因的上调使U2AF1基因S34F突变的SKM-1细胞增殖受到明显抑制、细胞凋亡率增加，细胞周期阻滞在S期和G2期；促炎因子IL-1β和TNF-α转录水平显著降低（P＜0.05），抑炎因子IL-4转录水平无明显变化（P＞0.05）.U2AF1基因S34F突变可诱导SKM-1细胞炎症反应表型，并通过FOXO3a-Bim信号通路在MDS中发挥作用.