<i>AHNAK</i>基因高表达与老年急性髓系白血病患者预后不良相关

doi:10.3877/cma.j.issn.2095-1221.2024.04.002

中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (04) : 204 -211. doi: 10.3877/cma.j.issn.2095-1221.2024.04.002

论著

AHNAK基因高表达与老年急性髓系白血病患者预后不良相关

杜鑫¹, 刘霞霞², 张恬波¹, 张夏林³, 杨林花⁴, 张睿娟¹^,^†(

)

^1. 030032　太原，山西医科大学第三医院　（山西白求恩医院　山西医学科学院　同济山西医院）血液内科
^2. 041000　临汾，山西省临汾市中心医院血液内科
^3. 030032　太原，山西白求恩医院　（山西医学科学院　同济山西医院），山西医科大学第三医院血液内科
^4. 030001　太原，山西医科大学第二医院血液内科

收稿日期:2024-02-12 出版日期:2024-08-01
通信作者: 张睿娟
基金资助:
山西白求恩人才引进基金(2021RC017)

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

Xin Du¹, XiaXia Liu², Tianbo Zhang¹, Xialin Zhang³, Linhua Yang⁴, Ruijuan Zhang¹^,^†()

^1. Department of Hematology, Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Taiyuan 030032, China
^2. Department of Hematology, Linfen Central Hospital, Linfen 041000, China
^3. Department of Hematology, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan 030032, China
^4. Department of Hematology, Second Hospital of Shanxi Medical University, Taiyuan 030001, China

Received:2024-02-12 Published:2024-08-01
Corresponding author: Ruijuan Zhang

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引用本文：

杜鑫, 刘霞霞, 张恬波, 张夏林, 杨林花, 张睿娟. AHNAK基因高表达与老年急性髓系白血病患者预后不良相关[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 204-211.

Xin Du, XiaXia Liu, Tianbo Zhang, Xialin Zhang, Linhua Yang, Ruijuan Zhang. The association between high AHNAK gene expression and poor prognosis in elderly AML patients[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(04): 204-211.

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。

关键词: 急性髓系白血病, 老年, 造血干细胞, 单细胞RNA测序, 预后

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.

Key words: Acute myeloid leukemia, Elderly, Hematopoietic stem cells, Single-cell RNA sequencing, Prognosis

表1 3例老年AML患者临床特点

患者	年龄（岁）	性别	骨髓原始细胞比例（%）	患者状况	染色体核型	基因突变
1	67	男	33	初诊	46, XY, del （20）（q4） [3] / 46, XY[7]	FLT3-ITD^low/NPM1
2	62	男	50	初诊	46, XY[20]	CEBPA^dm/GATA2
3	61	女	22.5	初诊	45, X, -X[1]	IDH2 /NPM1

表1 3例老年AML患者临床特点

患者	年龄（岁）	性别	骨髓原始细胞比例（%）	患者状况	染色体核型	基因突变
1	67	男	33	初诊	46, XY, del （20）（q4） [3] / 46, XY[7]	FLT3-ITD^low/NPM1
2	62	男	50	初诊	46, XY[20]	CEBPA^dm/GATA2
3	61	女	22.5	初诊	45, X, -X[1]	IDH2 /NPM1

表2 基因MALAT1、AHNAK、DMXL2和ATP8B4的引物序列

基因名称	引物序列（5'→3'）	预期扩增长度（bp）
MALAT1	上游AAAGCAAGGTCTCCCCACAAG	71
	下游GGTCTGTGCTAGATCAAAAGGCA
AHNAK	上游TGCCTGAGATTGCTACTGGTG	136
	下游CCTTTCAGTTTAGGAGACCCAAG
DMXL2	上游TGGAGGAGTATCAGAATTGGCT	109
	下游GCAGTATGTGCCTAGACAGTAAC
ATP8B4	上游ATAATCGGCAGTCTGAAGTGC	140
	下游TGGCTCACTACTTGATAGGAGAA
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

表2 基因MALAT1、AHNAK、DMXL2和ATP8B4的引物序列

基因名称	引物序列（5'→3'）	预期扩增长度（bp）
MALAT1	上游AAAGCAAGGTCTCCCCACAAG	71
	下游GGTCTGTGCTAGATCAAAAGGCA
AHNAK	上游TGCCTGAGATTGCTACTGGTG	136
	下游CCTTTCAGTTTAGGAGACCCAAG
DMXL2	上游TGGAGGAGTATCAGAATTGGCT	109
	下游GCAGTATGTGCCTAGACAGTAAC
ATP8B4	上游ATAATCGGCAGTCTGAAGTGC	140
	下游TGGCTCACTACTTGATAGGAGAA
β-actin	上游CATGTACGTTGCTATCCAGGC	250
	下游CTCCTTAATGTCACGCACGAT

图1 3例老年AML患者骨髓细胞scRNA-seq分析注：a图为3例老年AML患者骨髓细胞UMAP聚类分布情况，不同颜色代表不同的细胞类型；b图为热图展示各类型细胞的特异性高表达基因；c图为3例老年AML患者骨髓中不同细胞占比

图2 老年AML患者骨髓HSCs scRNA-seq分析注：a图为3例老年AML患者骨髓HSCs UMAP聚类分布情况，不同颜色代表不同HSCs亚群；b图为HSCs亚群CNV分析；c图为HSCs亚群干性分析；d、e图为HSCs拟时序分析，颜色越深代表衍化轨迹起点；HSCs各亚群拟时序分析，不同颜色代表不同的亚群细胞分化轨迹；f图为HSCs亚群RNA速率，不同颜色代表不同的HSCs亚群，箭头代表细胞分化方向；g图为HSCs亚群GSVA富集分析

图3 HSCs亚群差异基因表达分析注：a图为火山图展示HSCs _2、4和HSCs_1、3、5的差异基因表达，红点代表上调基因，蓝点代表下调基因；b图为差异表达基因KEGG富集通路

图4 差异表达基因MALAT1、AHNAK、DMXL2和ATP8B4的临床验证注：a~d为TCGA/GTEx数据库分析MALAT1、AHNAK、ATP8B4和DMXL2基因在AML患者和健康人中的表达，*为P<0.05，ns为P>0.05

图5 AHNAK基因在老年AML患者中的生存分析

表3 MALAT1、AHNAK、DMXL2和ATP8B4基因表达量（

± s）

基因	例数	MALAT1	AHNAK	DMXL2	ATP8B4
老年AML组	20	1.27 ± 0.23	4.28 ± 1.06	0.58 ± 0.07	0.64 ± 0.14
年轻AML组	20	0.45 ± 0.16^a	0.74 ± 0.14^a	1.37 ± 0.52	0.03 ± 0.01^a
健康对照组	20	1.04 ± 0.10	1.05 ± 0.11^a	1.05 ± 0.12	0.07 ± 0.02^a
F值		3.310	5.500	0.933	7.007
P值		0.060	0.020	0.415	0.010

表3 MALAT1、AHNAK、DMXL2和ATP8B4基因表达量（

± s）

基因	例数	MALAT1	AHNAK	DMXL2	ATP8B4
老年AML组	20	1.27 ± 0.23	4.28 ± 1.06	0.58 ± 0.07	0.64 ± 0.14
年轻AML组	20	0.45 ± 0.16^a	0.74 ± 0.14^a	1.37 ± 0.52	0.03 ± 0.01^a
健康对照组	20	1.04 ± 0.10	1.05 ± 0.11^a	1.05 ± 0.12	0.07 ± 0.02^a
F值		3.310	5.500	0.933	7.007
P值		0.060	0.020	0.415	0.010

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

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