HIF-1α Affects The Biological Function Of AML Cells Through TET2 Remodeling CXCR4 DNA Methylation

Acute myeloid leukemia is a type of malignant clonal disease originating from bone marrow hematopoietic cells that is refractory and has a high mortality rate^[7,8].Research confirms,the bone marrow hypoxic microenvironment enhances the recurrence and drug resistance of leukemia,and the hypoxic microenvironment activates hypoxia-inducible factors to promote the occurrence and development of AML.Recent studies have shown that the hypoxic microenvironment can regulate the occurrence and development of tumors by influencing epigenetic modifications.A large number of previous studies have confirmed that abnormal modification of genomic DNA methylation is a hallmark biological feature of AML^[81].However,there are few reports on the synergistic mechanism of hypoxic environment and epigenetic modification supporting the occurrence and development of AML.Therefore,this study aims to explore the mechanism by which hypoxia-inducible factor HIF-1αmediates DNA methylation to regulate the occurrence and development of AML,and to provide a theoretical basis for the treatment of leukemia.We first found that HIF-1α is highly expressed in human AML though analysising TCGA Cancer Genome Atlas data.Next,we select primary acute myeloid leukemia HL60 and NB4 cells for hypoxia（1%O₂）condition culture,and examine cell biological behavior.The results showed that HIF-1αprotein accumulated in HL60 and NB4 cells under hypoxic environment,and the proliferation of HL60 and NB4 cells are promoted,the migration of HL60 cells are increased,but the migration of NB4 cells did not change significantly.Based on the above results,we suppose that the expression of HIF-1αinduced by hypoxic environment is involved in regulating the biological behavior of AML cells.In order to prove the above speculation,we used Echinomycin,a specific inhibitor of HIF-1α,to inhibit the function of HIF-1αunder hypoxic and normoxic conditions.The results showed that the proliferation ability of HL60 and NB4 cells was significantly reduced,and the migration ability of HL60 cells was obviously reduced,while the apoptosis of HL60and NB4 cells did not change significantly.The above results further prove that the hypoxic microenvironment promotes the accumulation of HIF-1αto regulate the proliferation of HL60 and NB4 cells and the migration of HL60 cells.In order to reveal the mechanism by which HIF-1α regulates cell migration deeply,we used bioinformatics methods to analyze and select CXCR4,which is related to cell chemotaxis and migration and is significantly highly expressed in AML.We detected CXCR4 expression under hypoxic culture conditions,and the results showed that the CXCR4 transcription level was significantly up-regulated in HL60cells,but there was no significant change in NB4 cells.After knocking down CXCR4in HL60,it was found that the cell migration ability was significantly inhibited.Next,in order to prove the interaction between HIF-1αand CXCR4,we used the Ch IP-q PCR experimental method to verify.The results indicate that HIF-1αdirectly binds to the CXCR4 promoter.Therefore,we further speculate that the hypoxic microenvironment activates the accumulation of HIF-1αand up-regulates the expression of CXCR4 to drive the migration of HL60 cells.Studies have shown that HIF-1α enhances its transcriptional activity by binding to the hypoxia response element（HRE）in the promoter region of the DNA demethylase TET2 gene.Therefore,HIF-1αmay cause hypomethylation in the promoter region of CXCR4 gene by regulating TET2.In order to prove the above possibilities,we used the MSP method to verify the DNA methylation of the CXCR4promoter region in HL60 and NB4 cells.The results showed that the DNA methylation modification of the promoter region of CXCR4 gene in HL60 cells decreased under hypoxic culture conditions.At the same time,it was found that the expression of DNA demethylase TET2 was up-regulated under hypoxic environment but down-regulated after using Echinomycin to inhibit HIF-1α,and that TET2 was knocked down under hypoxic environment to up-regulate DNA methylation in the CXCR4 promoter region.In summary,our research proves that hypoxia activates the expression of HIF-1αand increases the expression of TET2 to reduce the DNA methylation level of the CXCR4 promoter,thereby promoting the binding of HIF-1αto the CXCR4 promoter and enhancing the migration ability of HL60 cells.