Investigation of the Roles of Interleukin-15 in T-cell Acute Lymphoblastic Leukemia
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摘要:
目的 初步探究白介素-15（interleukin-15，IL-15）在急性T淋巴细胞白血病（T-cell acute lymphoblastic leukemia，T-ALL）中的作用机制。 方法 利用公开发表的T-ALL患者数据集syn54032669（n = 1335 ）及GSE33315（n = 38）分析IL-15转录水平与患者生存情况及微小残留病（minimal residual disease，MRD）之间的关系；利用R语言中DESeq2包对IL-15高表达组与低表达组进行差异表达基因分析，并利用clusterProfiler等包对差异表达基因进行富集分析；Annexin V/7-AAD染色实验与细胞生长曲线检测IL-15对T-ALL细胞凋亡与生长的影响；实时荧光定量PCR与免疫印迹实验检测IL-15对PI3K/AKT通路的影响及其对下游基因的转录调控作用。结果 IL-15高表达组患者具有更长的总生存期（P < 0.05）与无事件生存期（P = 0.074），且MRD水平更低（P < 0.0001 ）；IL-15增加早期凋亡细胞比例（P <0.0001 ），但无法抑制T-ALL细胞的生长；IL-15显著下调神经营养受体酪氨酸激酶1（neurotrophic receptor tyrosine kinase 1，NTRK1）（P < 0.01）及成纤维细胞生长因子9（fibroblast growth factor 9，FGF9）（P < 0.05）的转录水平，并抑制NTRK1介导的PI3K/AKT通路激活；NTRK1与FGF9高表达组患者的临床预后更差（P < 0.05）。结论 IL-15在T-ALL中通过抑制NTRK1和FGF9的转录及NTRK1介导的PI3K/AKT通路激活发挥类似肿瘤抑制因子作用。 -
关键词:
- 急性T淋巴细胞白血病 /
- 白介素-15 /
- PI3K/AKT通路 /
- 临床预后
Abstract:Objective To preliminarily investigate the molecular mechanisms of interleukin-15 （IL-15） in T-cell acute lymphoblastic leukemia （T-ALL）. Methods Open-access T-ALL datasets syn54032669 （n = 1335 ） and GSE33315 （n = 38） were used to analyze the correlation between IL-15 levels and clinical features including survival and minimal residual disease （MRD）; DESeq2 package in R was used to identify differentially expressed genes between IL-15-high and IL-15-low groups; packages including clusterProfiler were utilized to perform enrichment analyses; Annexin V/7-AAD staining and cell growth curves were performed to analyze the effects of IL-15 on the apoptosis and growth of T-ALL cells; real-time quantitative PCR and Western blot were performed to validate the effects of IL-15 on PI3K/AKT pathway and transcription of downstream genes.Results T-ALL patients with high IL-15 levels had longer overall survival （P < 0.05） and event-free survival （P = 0.074） but lower MRD levels （P < 0.0001 ）; IL-15 increased the proportion of early apoptotic cells but failed to inhibit the growth of T-ALL cells; IL-15 remarkably inhibited the transcription of neurotrophic receptor tyrosine kinase 1 （NTRK1） （P < 0.01） and fibroblast growth factor 9 （FGF9） （P < 0.05）, and also NTRK1-mediated activation of PI3K/AKT pathway; T-ALL patients with high NTRK1 and FGF9 levels had worse prognosis （P < 0.05）.Conclusion IL-15 exerts tumor suppressor-like functions by repressing the transcription of NTRK1 and FGF9, as well as inhibiting PI3K-AKT pathway activated by NTRK1 in T-ALL. -
图 1 IL-15表达水平与患者预后之间的相关性（$\bar x \pm s $）
A：IL-15高表达组与低表达组的OS（左）及EFS（右）曲线；B：IL-15高表达组与低表达组中MRD水平的变化；C：治疗第46天MRD阳性组与阴性组中IL-15表达量的变化；D：不同亚型T-ALL患者中IL-15表达量的变化；E：IL-15高表达组与低表达组的免疫浸润分析结果；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 1. Correlation between IL-15 level and clinical prognosis （$\bar x \pm s $）
图 2 IL-15高表达组与低表达组之间DEGs的火山图
Figure 2. Volcano plot of DEGs between IL-15-high and IL-15-low groups
图 3 富集分析结果
A：上调DEGs的KEGG富集分析；B：NOTCH信号通路（左）以及PI3K/AKT通路（右）的GSEA富集分析；NES（normalized enrichment score）：归一化富集得分。
Figure 3. Results of enrichment analyses
图 4 免疫印迹实验检测IL-15处理后p-AKT的蛋白水平变化
Figure 4. Western blot assay of changes in p-AKT level after IL-15 treatment
图 5 IL-15对T-ALL细胞生长与凋亡的影响（$\bar x \pm s $，n = 3）
A：柱状图显示各凋亡阶段比例及统计学分析结果；B：AnnexinV/7-ADD染色检测细胞凋亡情况；C：细胞生长曲线显示IL-15处理后T-ALL细胞的生长速度变化；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 5. Influences of IL-15 on the growth and apoptosis of T-ALL cells （$\bar x \pm s $，n = 3）
图 6 IL-15对HSCs相关基因以及T细胞发育阻滞的影响（$\bar x \pm s $，n =
1068 ）A：HSCs上调基因集（左）以及下调基因集（右）的GSEA分析结果；B：IL-15高表达组与低表达组中ETP分数（左）、双阴性早期阶段分数（中）及双阳性晚期阶段分数（右）的变化；C：IL-15高表达组与低表达组中KIT转录水平的变化；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 6. Influences of IL-15 on HSCs-related genes and developmental retardation of T cells （$\bar x \pm s $，n =
1068 ）
图 7 IL-15与NTRK1以及FGF9的表达相关性分析（$\bar x \pm s $，n = 3）
A：HSCs与PI3K/AKT通路相关基因的韦恩图；B：IL-15与NTRK1（左）或FGF9（右）的表达量散点图；C：RT-qPCR检测IL-15处理20 h后NTRK1与FGF9表达量的变化；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 7. Correlations between IL-15 and NTRK1 or FGF9 （$\bar x \pm s $，n = 3）
图 8 NTRK1对PI3K/AKT通路以及T细胞发育阻滞的影响（$\bar x \pm s $，n =
1335 ）A：免疫印迹实验检测larotrectinib处理24 h后p-AKT的蛋白水平变化；B：NTRK1高表达组与低表达组中ETP分数（左）以及双阳性晚期阶段分数（右）的变化；nsP > 0.05；*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 8. Influences of NTRK1 on PI3K/AKT pathway and developmental retardation of T cells （$\bar x \pm s $，n =
1335 ）
图 9 NTRK1或FGF9的表达水平对T-ALL患者生存的影响
A：NTRK1高表达组与低表达组的OS曲线；B：NTRK1高表达组与低表达组的EFS曲线；C：FGF9高表达组与低表达组的OS曲线；D：FGF9高表达组与低表达组的EFS曲线。
Figure 9. Influences of NTRK1 or FGF9 levels on patients’ survival
图 10 基于NTRK1或FGF9表达量进行生存分析的ROC曲线
A：基于NTRK1表达量进行OS分析的ROC曲线；B：基于NTRK1表达量进行EFS分析的ROC曲线；C：基于FGF9表达量进行OS分析的ROC曲线；D：基于FGF9表达量进行EFS分析的ROC曲线；以上OS与EFS分析均选取半年、三年与十年时间点；图上标注约登指数最大化时的灵敏度以及特异度。
Figure 10. ROC plots of survival analyses based on NTRK1 or FGF9 levels
图 11 NTRK1或FGF9的表达上调提示不良预后（$\bar x \pm s $）
A：NTRK1（左）或FGF9（右）高表达组与低表达组中MRD水平的变化；B：MRD结果不同的患者中NTRK1（左）及FGF9（右）表达量的变化；C：不同亚型T-ALL患者中NTRK1（左）及FGF9（右）表达量的变化； nsP > 0.05，*P < 0.05；**P < 0.01；***P < 0.001；****P < 0.0001。
Figure 11. Elevated NTRK1 or FGF9 levels predict worse prognosis （$\bar x \pm s $）
表 1 实时荧光定量PCR使用的引物序列
Table 1. Primer sequences designed for real-time quantitative PCR
基因 引物序列（5’-3’） 引物长度（bp） NTRK1 F: GCTGGCTCTTCAATGGCTC 19 R: GTGTAGTTGCCGTTGTTGACG 21 FGF9 F: TGCAGGACTGGATTTCACTTAGA 23 R: ACTCTTGGGTTAGTTTTTCTGATCC 24 ACTB F: GTTGAGAACCGTGTACCATGT 21 R: TTCCCACAATTTGGCAAGAGC 21 
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