Arsenic Trioxide For The Regulation Of WNT Pathway Inhibiting Genes SFRPs DNA Methylation And Its Effects On The Gene Expression In Acute Promyelocytic Leukemia Cell Line NB4

Objective: To study the influence of arsenic trioxide on the expression of WNT signaling pathway inhibiting factor SFRPs(secreted frizzle related proteins) in acute promyelocytic leukemia cells; To explore its impact on the genes promoter region methylation condition,in order to further ascertain As2O3 possibly potential epigenetic regulation mechanisms to the treatment of APL that may exist. Methods: 1.In order to explore the molecular mechanism of apoptosis caused by As2O3 preliminarily, CCK-8 method was used to detect As2O3 effects on cell proliferation,and used flow cytometry to detect cell apoptosis and cell cycle changes and then detected cell apoptosis related proteins expression levels with Western blot method After using arsenic trioxide to act on the acute promyelocytic leukemia cell line NB4 with the logarithmic growth phase; 2. The technology of BSP and TA cloning and sequencing was used to detect the control group(not dosing group) and the dosing group cells SFRPs(SFRP1 SFRP2, SFRP4, SFRP5) promoter CpG island methylation changes, and analysis of the correlation between SFRPs promoter region methylation level and cell apoptosis to explore As2O3 possibly potential epigenetic changes to the treatment of APL;And through the Real Time-qPCR and Western blot methods detecting related DNMTs(DNA methyltransferases) expression level changes, to analye the cause of cellular epigenetic changes related mechanisms by As2O3.3. Through the Real Time- qPCR method detect the cells WNT signal pathway inhibitory factor(SFRP1 SFRP2, SFRP4, SFRP5) expression level changes before and after drugs, analyze its correlation with cell apoptosis after drug; Using the Real Time-qPCR method to detect the expression changes of WNT signal pathway key molecular beta-catenin and downstream target genes Survivin etc, to further explore As2O3 related molecular mechanism to the treatment of APL. Results: 1. The arsenic trioxide can inhibit the proliferation of NB4 cells, and promote cell apoptosis, andhave drug concentration dependence; As2O3 can promote the expression of apoptosis related proteins.2. Through the detection find that no medicine group NB4 cells SFRPs(SFRP1 SFRP2, SFRP4, SFRP5) promoter regions have varying degrees of hypermethylation, SFRP1/4 promoter region methylation rate decreased after As2O3 treatment, and negatively correlated with cell apoptosis, and SFRP2/5 promoter region methylation rate down is not obvious;The control group compared with dosing group, DNMTs(DNMT1, DNMT3 A and DNMT3B) protein expression decreased,and DNMT1 downregulation is negatively correlated with drug concentration.3. After the treatment of As2O3, NB4 cells WNT signal pathway inhibiting factor SFRP1,SFRP4 significantly upregulated;SFRP2, SFRP5 expression upregulated is not obvious, and the dosing group compared with the control group, WNT signaling pathways key downstream genes expression reduced; SFRP1/4 expression upregulation and cell apoptosis rate present positive correlation.Conclusions: Arsenic trioxide can inhibit the proliferation of NB4 cells and promoting apoptosis, may be related to reversing the methylation of WNT signaling pathways SFRPs raised their protein expression, to inhibit the excessive activation of WNT signaling pathway, and arsenic agent to methylation may be associated with downregulation DNMTs expression.The molecular mechanism of arsenic agent in the treatment of APL is complex, in addition to targeted degradation of PML-RAR alpha fusion gene,however epigenetic regulation function of arsenic agent may also be one of the important molecular mechanism.The epigenetic regulation of targeted drugs, are expected to become a new direction in the treatment of APL.