Effect Of Arsenic Trioxide On Gene Expression Of Human Acute Myeloid Leukemia Cells(KG-1a) Based On RNA-Seq Technology

BackgroundAcute myeloid leukemia(AML)is a highly heterogeneous malignant tumor with high mortality and recurrence rate.The reason for relapse is the existence of stem cells in leukemia patients.Arsenic trioxide is a multi-target chemotherapy drug.Previous studies have found that arsenic trioxide can trigger the inhibition of cell proliferation of KG-1a cell line which is an acute myeloid leukemia cell line with leukemia stem cell characteristics,but the specific mechanism is not clear.RNA-Seq analysis has the following characteristics,such as a large amount of data,quantitative accuracy and high repeatability.Therefore,it is significant to study the gene expression profile changes of KG-1a cells caused by arsenic trioxide.ObjectiveThe objective of this study was to study the effects of arsenic trioxide(ATO)on the proliferation and apoptosis of acute myeloid leukemia KG-1a cells,to explore the response genes closely related to arsenic trioxide based on high-throughput sequencing technology,and to provide experimental basis for elucidating the mechanism of arsenic trioxide.Methods1.The effect of ATO on KG-1a cell proliferation was detected by CCK-8 method.2.The effect of ATO on KG-1a cell apoptosis was detected by flow cytometry.3.The effect of ATO on KG-1a cell cycle was detected by flow cytometry.4.Differential genes(DEGs)between ATO 2 μmol/L group and control group were obtained by transcriptome sequencing.5.GO and KEGG were used for enrichment analysis.6.Use Cytoscape and String to construct DEGs protein interaction network in order to screen Hub-genes.7.Q-PCR was used to verify the m RNA expression.8.Western blot was used to verify the protein expression.Results1.The results of CCK-8 showed that when ATO concentration was less than 3 μmol/L,and the difference of inhibition rate between different concentration groups was statistically significant(P<0.05).2.The results of flow cytometry showed that when the concentration of ATO was between 1.5 μmol/L and 3 μmol/L,the apoptosis rate of the treatment group was significantly higher than that of the control group(P<0.05).3.The results of flow cytometry showed that when the concentration of ATO was less than 2 μmol/L,the distribution rate of G2/M phase cells in different concentration groups was significantly different from that in the control group(P<0.05).4.RNA-Seq showed that there were 5371 differentially expressed genes,of which 2666 is up-regulated and 2705 is down-regulated.5.Go enrichment results showed that the biological process items enriched by downregulated differential genes were mainly related to ribonucleic acid-protein complex biogenesis,DNA replication,DNA biosynthesis,methylation,cell cycle G1/S phase transition,and telomere maintenance through telomerase and the up-regulated terms enriched mainly involved in response to unfolded proteins and topological error proteins,granulocyte activation and mediated immune response,detoxification process,autophagy and autophagy regulation and other processes.6.KEGG enrichment results show that the down-regulated differential genes are mainly involved in ribosome production in eukaryotes,purine and pyrimidine metabolism,RNA transport,cell cycle,spliceosome,human T cell leukemia virus type I infection,DNA replication,RNA polymerase,TGF-β pathway and other related pathways;the up-regulated differential genes are mainly involved in the lysosome,peroxisome,phagosome,glutathione metabolism,cytochrome P450 metabolism of foreign substances,autophagy,chemical carcinogenesis function,amino sugar and nucleotide sugar metabolism,mineral absorption,PPAR pathway and other related signal pathways.7.PPI interaction network results showed that the core network node genes were MYC,MCM7 and PCNA.8.The results of real-time PCR showed that the m RNA expression levels of different genes(MYC,MCM7,PCNA,BCL2L1,CCNE1,BAX,BAD,CDKN1 A,CDKN1C and CDKN3)in KG-1a cell and other three different AML cell lines were consistent with the result of RNA-seq.9.The results of Western blot showed that the protein expression levels of MYC,MCM7,PCNA,BCL2L1,BAX,BAD,P21 and P57 protein in KG-1a cell and other three different AML cell lines were consistent with the result of RNA-seq.ConclusionArsenic trioxide can induce the proliferation inhibition,apoptosis and cell cycle arrest of KG-1a cells.RNA-Seq analysis showed that the pathways related to DNA replication,apoptosis and cell cycle regulation were significantly enriched,among which MYC,PCNA and MCM7 may play key roles.This study provides a reliable clue for further improving the therapeutic effect of ATO on AML.