Molecular Mechanism Of Arsenic Trioxide In The Treatment Of Acute Promyelocytic Leukemia Based On High-Throughput Sequencing

Acute promyelocytic leukemia(APL)is characterized by a large number of abnormal granular myeloblasts in peripheral blood and bone marrow,with severe bleeding tendency and poor prognosis.Arsenic trioxide(ATO)is the most important active ingredient in the traditional Chinese medicine arsenic,which belongs to the highly toxic mineral medicine.Arsenic is hot in nature,bitter and sour in taste,and highly toxic.Modern studies have found that ATO can be used not only for the treatment of many solid tumors,but also for leukemia.However,the molecular mechanism of ATO in the treatment of leukemia is not completely clear.In this study,It is the first time to screen the key genes and signal pathways that inhibit the activity of HL-60 cell lines by ATO through RNA-Seq sequencing,to further explore the molecular mechanism of ATO in APL treatment and its effect on cell proliferation,cell cycle,and apoptosis.To provide reference for the development of new anti APL drugs and the identification of effective targets for APL molecular targeted therapy.In this study,CCK-8 assay,flow cytometry and Western blot were used to investigate the effect of ATO on the activity of HL-60 cells.The key DEGs and signaling pathways of ATO in APL treatment were screened by RNA-Seq sequencing and verified by Western blot.The results were as follows:1.CCK-8 assay showed that ATO significantly inhibited the proliferation of HL-60 cells in a concentration-dependent manner,with an IC50 value of 4.2 μmol/L.The result of flow cytometry showed that different concentration of ATO could promote apoptosis and arrest cell cycle in G2/M phase of HL-60 cells after 24 h treatment.Western blot showed that ATO could significantly down-regulate Bcl-2 expression and up-regulate Bax/Bcl-2 ratio,and induce apoptosis of HL-60 cells.The results of Western blot showed that ATO could significantly decrease the expression of Bcl-2 protein and increase the ratio of Bax/Bcl-2,thus promoting apoptosis of HL-60 cells;2.Analysis of RNA-seq sequencing data showed that using | log2 Fold change | ≥1 and q<0.05 as thresholds,the expression of DEGs was analyzed between the control group and the dosing group.4550 DEGs were selected,of which 1174 were up-regulated and 3376 were down-regulated;3.GO enrichment results showed that there wre 17 terms related to cellular components,24 terms related to biological processes,and 12 terms related to molecular functions.KEGG analysis showed that the PI3K/AKT signaling pathway,cytokine receptor interaction,MAPK signaling pathway,Rapl signaling pathway and so on enriched more DEGs.These results suggest that ATO can regulate the PI3K/AKT signaling pathway in HL-60 cells and affect cell proliferation and apoptosis;4.Ten key node genes including TNF,STAT3,IL1B,ITGAM,CXCL8,ICAM1,VEGFA,JUN,TGFB1 and SRC were identified by PPI interaction network analysis;5.Western blot analysis showed that the expression levels of TNF,STAT3 and IL1B decreased,which was consistent with the results of sequencing.The results showed that the expression of PI3K and AKT had no significant change,but the expression of p-PI3K and pAKT decreased significantly.Conclusion:In this study,we investigated the effects of ATO on the proliferation,cell cycle and apoptosis of HL-60 cells.The data showed that ATO could significantly inhibit the proliferation of HL-60 cells and induce G2/M arrest.It can increase the expression of Bax protein and decrease the expression of Bcl-2.According to the results of RNA-seq sequencing,functional enrichment analysis of DEGs and protein interaction network analysis,the key DEGs of ATO inhibiting HL-60 cell activity were identified as TNF,STAT3 and IL1B.It was suggested that PI3K/AKT pathway played an important role in the process of ATO inhibiting HL-60 cell proliferation and inducing apoptosis.The results of protein verification were consistent with the sequencing results.It was shown that ATO could down-regulate the protein expression of TNF,STAT3 and IL1B in HL-60 cells,but had no significant effect on the protein expression of PI3K and AKT,but could significantly down-regulate the protein expression of p-PI3K and p-AKT.