| Objective:A bioinformatics approach was used to construct a prognostic model of acute myeloid leukaemia based on cuproptosis and to preliminarily explore the molecular function of one of the model genes,PACS2,in HL-60 of acute myeloid leukemia cells.Methods:(1)The TCGA database and GTEx database were used to download transcriptome sequencing data containing 173 AML patients and 70 normal bone marrow samples to obtain differentially expressed genes.(2)The cuproptosis score of 173 AML patients was calculated as a trait for weighted gene co-expression network analysis,and then highly correlated modules were obtained to screen out the hub genes.(3)The differentially expressed gene was intersected with the hub gene to obtain the core gene,and the core gene was analyzed for functional enrichment.(4)Univariate Cox regression analysis and Lasso regression analysis screened out two model genes,NDUFV1 and PACS2,and then constructed a risk model.Divide the training,internal,and external validation sets into high-risk and low-risk groups.The survival curve shows lower survival rates for patients in the high-risk group.The area under the curve for1,3 and 5 years in the ROC curve is greater than 0.6,indicating that the established risk model is reliable.(5)Cox regression analysis was used to perform independent prognostic analysis,and nomogram was drawn according to the independent prognostic factors screened.Finally,the function and pathway of model genes were explored by single-sample gene set enrichment analysis.(6)RT-q PCR was used to detect the expression of model genes in patients with acute myeloid leukemia and normal human specimens(7)Acute myeloid leukemia cell line HL-60 was cultured in vitro.(8)Cell transfection was performed by cell transfection technology,and the silencing effect of PACS2 gene was detected by RT-q PCR and Western Blot.(9)The effects of PACS2 on proliferation and apoptosis of HL-60 cells were investigated by CCK-8 experiment and TUNEL experiment.(10)Western Blot experiments were used to preliminarily explore the changes of related protein molecules involved in the influence of PACS2 on HL-60 cell proliferation.Results:(1)Through the analysis of transcriptome sequencing data of AML patients and normal people,11160 differentially expressed genes were obtained.(2)The weighted gene co-expression network analysis was aggregated into 8 modules,among which the MEyellow module showed the most correlation with cuproptosis score,and 132 hub genes were screened.(3)11160 differential expressed genes and 132 hub genes intersected,resulting in a total of 32 core genes.It is mainly enriched into functions such as cytoplasmic microtubule tissue and RNA methylation,as well as m TOR signaling pathway and Notch signaling pathway in tumors.(4)Two model genes,PACS2 and NDUFV1,were identified by univariate Cox and Lasso analysis.Survival curve showed that the survival rate of high-risk patients was lower,and the area under the curve of 1-,3-and 5-year in ROC curve were all greater than 0.6,indicating that the established risk model was reliable.(5)Cox independent prognostic analysis screened age and risk score as independent prognostic factors for AML.The C-index for plotting the nomogram is 0.658.It predicts the likely 1-,3-,and 5-year survival rates in patients with AML.The results of singlesample gene set enrichment analysis showed that PACS2 genes were mainly rich in phosphoinositide metabolism and histone modification.NDUFV1 is mainly rich in nc RNA metabolism,carbon metabolism,2-oxocarboxylic acid metabolism and other pathways.(6)The results of RT-q PCR showed that compared with the normal control group,the expression of PACS2 in patients with acute myeloid leukemia was significantly increased(P=0.0038),and the expression of NDUFV1 was not statistically significant(P>0.05)(7)After plasmid transfection of HL-60 cells,RT-q PCR and Western Blot experiments showed that si-PACS2-3 had the most significant silencing effect compared with the si-NC group(P=0.0070).(8)The results of CCK-8 and TUNEL experiments showed that the proliferation of HL-60 cells in the si-PACS2-3 group was significantly inhibited(P<0.0001)and the apoptosis level was significantly increased(P<0.0001)compared with the si-NC group.(9)The results of Western Blot experiments showed that after silencing PACS2,the level of PTEN protein increased,the level of p-AKT protein decreased.Conclusion:(1)The prognostic model of acute myeloid leukemia based on cuproptosis was successfully constructed and validated by bioinformatics methods,and model genes including PACS2 and NDUFV1 were potential prognostic markers for acute myeloid leukemia.(2)PACS2 is highly expressed in patients with acute myeloid leukemia.(3)Silencing PACS2 inhibits HL-60 cell proliferation and promotes apoptosis.(4)PACS2 may inhibit the proliferation of HL-60 in leukemia cells by affecting the expression of PTEN and p-AKT protein molecules. |
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