AHNAK基因高表达与老年急性髓系白血病患者预后不良相关

The association between high AHNAK gene expression and poor prognosis in elderly AML patients

目的基于单细胞RNA测序(scRNA-seq)筛选老年急性髓系白血病(AML)造血干细胞(HSCs)中的关键基因,分析其对预后的影响。方法对3例初诊老年AML患者骨髓细胞进行scRNA-seq,采用统一流形逼近与投影(UMAP)算法聚类确定骨髓细胞类型。进一步根据拷贝数变异(CNV)分析、干性分析、拟时序分析、差异表达基因分析和富集分析揭示HSCs亚群的特点,筛选差异表达基因。通过癌症基因组图谱计划(TCGA)数据库/基因型-组织表达(GTEx)数据库以及实时荧光定量聚合酶链反应(RT-qPCR)对收集的老年AML患者、年轻AML患者和健康人骨髓样本检测,对筛选出的关键基因表达进行验证。采用单因素方差分析和Dunnett-t检验进行比较,生存分析用Log-rank检验。结果3例老年AML患者的骨髓细胞UMAP聚类分为11类细胞,其中HSCs比例差异较大;重新UMAP聚类HSCs,得到5个HSCs亚群。HSCs_2和4在细胞命运2(Fate2)分支,CNV评分较高,且富集通路类似,因此归为一组,HSCs_1、3和5归为另一组;差异表达基因分析结果显示肺腺癌转移相关转录本1(MALAT1)、AHNAK核蛋白(AHNAK)、Dmx like蛋白2(DMXL2)和磷脂转运ATP酶8B4(ATP8B4)基因差异显著。TCGA数据库/GTEx数据库分析结果显示,与健康人比较,MALAT1、AHNAK和ATP8B4基因在AML患者中高表达(P<0.05),DMXL2基因表达在AML患者和健康人中比较差异无统计学意义。RT-qPCR结果显示AHNAK和ATP8B4 mRNA水平在老年AML患者中高于年轻AML患者和健康人(P<0.05)[AHNAK mRNA:(0.74±0.14)比(4.28±1.06),(1.05±0.11)比(4.28±1.06),ATP8B4 mRNA:(0.03±0.01)比(0.64±0.14),(0.07±0.02)比(0.64±0.14)]。MALAT1 mRNA水平在老年AML患者中高于年轻AML患者[(0.45±0.16)比(1.27±0.23)](P<0.05),但与健康人的差异无统计学意义。DMXL2 mRNA水平在老年AML患者中与年轻AML患者、健康人比较差异无统计学意义。生存分析显示AHNAK基因高表达与老年AML患者生存负相关。结论AHNAK基因在老年AML患者HSCs中高表达,且与预后不良相关。

Objective To screen the key genes in hematopoietic stem cells(HSCs)from elderly acute myeloid leukemia(AML)patients using single-cell RNA sequencing(scRNA-seq)and analyze their prognostic impact.Methods Bone marrow cells from three newly diagnosed elderly AML patients were subjected to scRNA-seq.Cell types were classified by uniform manifold approximation and projection(UMAP)clustering algorithm.Copy number variation(CNV)analysis,stemness analysis,pseudotime analysis,differential gene expression analysis,and enrichment analysis was used to identify characteristic genes of HSCs subgroups.The expression of selected key genes was validated using The Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)databases,as well as real-time quantitative polymerase chain reaction(RT-qPCR)on collected bone marrow samples from elderly AML patients,young AML patients,and healthy individuals.Univariate ANOVA and Dunnett-t test were used for comparison the differences among groups.Survival analysis was analysed by Log-rank test.Results Eleven cell types were identified in three elderly AML patients,with significant differences in the proportion of HSCs.Five subsets of HSCs were identified after reclustering.HSCs_2 and HSCs_4 showed higher CNV scores and similar enrichment pathways,which were in the branch of cell fate 2(Fate2),thus being grouped into a separate category from subsets HSCs_1,HSCs_3,and HSCs_5.Differential expression gene analysis highlighted that metastasis associated in lung denocarcinoma transcript 1(MALAT1),AHNAK nucleoprotein(AHNAK),Dmx like 2(DMXL2)and ATPase phospholipid transporting 8B4(ATP8B4)genes were significantly differentially expressed.Analysis of TCGA and GTEx databases confirmed high expression levels of MALAT1,AHNAK,and ATP8B4 genes in AML patients,which significantly different from healthy individuals(P<0.05),but without significant difference in the expression of DMXL2.RT-qPCR results showed that AHNAK and ATP8B4 mRNA expressions were significantly higher in elderly AML patients compared to young AML patients and healthy individuals[AHNAK mRNA:(0.74±0.14)vs(4.28±1.06),(1.05±0.1)vs(4.28±1.06),ATP8B4 mRNA:(0.03±0.01)vs(0.64±0.14),(0.07±0.02)vs(0.64±0.14)](P<0.05),while MALAT1 mRNA levels in elderly AML patients were significantly higher compared to young AML patients[(0.45±0.16)vs(1.27±0.23)](P<0.05),but no significant difference compared to healthy individuals.No significant difference was observed in the expression of DMXL2 mRNA.Survival analysis showed that elderly AML patients with high AHNAK expression had poor prognosis.Conclusions AHNAK was highly expressed in HSCs of elderly AML patients and was associated with poor prognosis.