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

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

目的: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。方法: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。结果: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)。结论: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。

Objective: To investigate the effect of U2 AF1 gene mutation to inflammatory cytokine in SKM-1 cell of human myelodysplastic syndromes( MDS),and whether the above effects were mediated by FOXO3 a-Bim signaling pathway. Methods: Wide-type U2 AF1 and mutant U2 AF1( the serine residue 34 was replaced by phenylalanine,and named as S34 F) recombinant expression plasmids were constructed. Lentiviruses were packaged and transfected into SKM-1 cells. The expression of FOXO3 a 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. FOXO3 a,Bim,Bcl-2 and Bax protein expression levels were detected by Western blot. Results: Compared with the control group,the cell apoptosis rate,pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly increased in the S34 F 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 FOXO3 a,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 FOXO3 a could significantly inhibited the proliferation and increased cell apoptosis of SKM-1 cells with U2 AF1 S34 F mutation;cell cycle was blocked at the S and G2 phases;the proinflammatory 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). Conclusion: U2 AF1 S34 F mutation can regulate inflammatory phenotype in SKM-1 cells,which may be mediated through FOXO3 a-Bim signaling pathway.