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

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

Abstract

Abstract:

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

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]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(04): 204-211.

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References 29

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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

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

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

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

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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