Research On The Mechanism Of Active DNA Demethylation In Myelodysplastic Syndrome By The Active Ingredients Of Qing Huang Powder

PART Ⅰ:Research on the Active Demethylation Effect of Qinghuang Powder on MDS Based on TET2Objectives:To explore the mechanism by which the active ingredients of Qinghuang powder,including sulfide arsenic and indirubin carmine,act on MDS cell lines through TET2-mediated active demethylation.Methods:The MDS cell line SKM-1 was used in this study.Different concentrations of sulfide arsenic and indirubin carmine were used to intervene in the cells,and the cell viability of SKM-1 cells was detected using the CCK8 assay.The changes in 5-hydroxymethylcytosine(5-hmC)and 5-methylcytosine(5mC)were detected using the DNA dot blot method.The methylation levels of CpG sites in the promoter regions of the PIK3R2 and PTPN6 genes in SKM-1 cells were detected using bisulfite sequencing,and the mRNA and protein levels were verified.The mRNA and protein expression of TET2 in SKM-1 cells were detected using real-time quantitative PCR and protein immunoblotting.The effect of sulfide arsenic intervention on the expression of the PI3K/AKT signaling pathway in SKM-1 cells was detected using protein immunoblotting.The apoptosis of SKM-1 cells after sulfide arsenic,indigo carmine,and the PI3K/AKT pathway inhibitor LY294002 intervention was detected using flow cytometry.Results:(1)CCK8 proliferation assay:Arsenic sulfide inhibited the activity of SKM-1 cells,and this inhibition became more significant with increasing concentration(P<0.05).Indirubin did not show significant inhibitory effects on SKM-1 cell viability.(2)DNA dot blot experiment:Both arsenic sulfide and indirubin increased the expression of 5hmc and decreased the expression of 5mC in SKM-1 cells(P<0.05).The effect was most significant at a concentration of 2 μM for arsenic sulfide and 20 μM for indirubin(P<0.05).(3)Methylation detection and validation experiment:Arsenic sulfide decreased the methylation level of PIK3R2(P<0.05),but indirubin had no significant effect on PIK3R2 methylation.Both arsenic sulfide and indirubin had no significant effect on PTPN6 methylation.RT-PCR validation experiment showed that arsenic sulfide promoted the mRNA and protein expression levels of PIK3R2(P<0.05),while indirubin significantly inhibited the mRNA expression level of PIK3R2(P<0.05),but had no significant effect on the protein expression level of PIK3R2.(4)Effect of Qinghuang powder on TET2,P-PI3K and P-AKT expression in MDS cells:Arsenic sulfide increased the mRNA expression level of TET2(P<0.05),while indirubin had no effect on TET2 mRNA expression.At the protein level,arsenic sulfide significantly inhibited the expression of TET2 protein(P<0.05),while indirubin significantly promoted the expression of TET2 protein(P<0.05).Arsenic sulfide significantly inhibited the protein expression levels of P-PI3K and P-AKT(P<0.05),but had no significant effect on AKT protein expression levels.(5)Effect of Qinghuang powder on apoptosis of MDS cells:Arsenic sulfide significantly promoted the apoptosis of SKM-1 cells(P<0.05),while indigo naturalis had no significant effect on SKM-1 cell apoptosis(P>0.05).LY294002 effectively promoted the apoptosis of SKM-1 cells(P<0.05).Conclusion:(1)Both arsenic sulfide and indigo carmine,the effective components in Qinghuang powder,have demethylation effects.(2)Indigo carmine significantly promotes the expression of TET2 protein,which is related to active DNA demethylation;arsenic sulfide has no effect on the expression level of TET2 protein.(3)Arsenic sulfide can promote SKM-1 cell apoptosis by inhibiting the PI3K/AKT signaling pathway.Part Ⅱ:High-throughput sequencing to screen candidate miRNAs involved in the promotion of TET2 expression by Indirubin in myelodysplastic syndromeObjective:To screen candidate miRNAs involved in the promotion of TET2 expression by Indirubin in myelodysplastic syndrome.Methods:(1)The MDS cell line SKM-1 was used as the research object.The cells were treated with 20μM Indirubin for 48 hours,and high-throughput sequencing was used to detect the microRNAs in the cells.The TPM(Transcript Reads Number Per Million)method was used for data normalization,and DESeq R was used for differential expression analysis between the two groups to screen for differentially expressed microRNAs.(2)Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze the functions and utilities of the differentially expressed miRNA candidate target genes.(3)The miRanda software was used for microRNA-TET2 target gene prediction analysis to screen for candidate microRNAs that may regulate the TET2 gene.(4)The differentially expressed microRNAs were verified by real-time quantitative PCR using the MDS cell line SKM-1 as the research object and treated with 20μM Indirubin for 48 hours.Results:(1)Effect of Indigo Carmine on miRNA expression in MDS cellsThe study compared the differences in miRNA expression between the control group and the indigo carmine group in SKM-1 cells using high-throughput sequencing.It was found that there were significant differences in the expression levels of 35 miRNAs between the two groups.Compared with the control group,17 miRNAs were up-regulated and 18 miRNAs were down-regulated in the indigo carmine group.(2)Functions of differentially expressed miRNAsGO functional enrichment analysis found that the down-regulated miRNA candidates targeted by indigo carmine were mainly involved in biological processes such as metal ion transport,nucleotide excision repair,and biological stimulus response,while the up-regulated miRNA candidates were mainly involved in biological processes such as cell differentiation,intracellular signaling,and peptidyl-proline modification.KEGG pathway enrichment analysis found that the down-regulated miRNA candidates were mainly involved in metabolic processes and signaling pathways such as carbon metabolism,fructose and mannose metabolism,galactose metabolism,and viral carcinogenesis,while the up-regulated miRNA candidates were mainly involved in signaling pathways related to cancer such as small cell lung cancer,pancreatic cancer,gastric cancer,cancer signaling pathways,and chronic myeloid leukemia.(3)Prediction and verification of candidate microRNAs regulating TET2 geneThrough bioinformatics analysis,miRNAs with significant differential expression were predicted for their target genes.It was found that miRNA-29b-3p and miRNA125a-5p might regulate TET2.RT-PCR experiments verified that indigo carmine could down-regulate the expression of miRNA-29b-3p and up-regulate the expression of miRNA-125a-5p,which was consistent with the miRNA sequencing results.Conclusion:(1)Indirubin can significantly affect miRNA expression in MDS cells.(2)Indirubin may promote the expression of TET2 by inhibiting the expression of miRNA-29b-3p.