Screening New Biomarkers In Acute Myeloid Leukemia And Study Of Their Mechanism

Introduction: Acute myeloid leukemia(AML)is a haematopoietic neoplasm characterized by clonal proliferation,differentiation blockade and inhibited apoptosis of malignant stem or progenitor cells.AML has poor clinical outcomes and a high mortality rate,and its overall five-year survival rate is less than 50%.Standard therapies often fail to achieve complete remission(CR),and relapse may occur after CR.Therefore,it is necessary for us to further explore the internal mechanism of the initiation and development of AML and find new molecular markers to provide effective treatment and prognosis prediction strategies.Methods:(1)Database information and tissue specimens: The data used for bioinformatics analysis is obtained from TCGA,VIZOME,GEO and GDSC data platform.We selected the TCGA(The Cancer Genome Atlas)data platform as the main analysis database and selected other data platform serves as the associate and external verification database.The tissue specimens required for molecular biology experiment verification were obtained by our department with the informed consent of the patients and their family members.The relevant experimental plan was reviewed and approved by the ethics committee of the First Affiliated Hospital of China Medical University.(2)Bioinformatics analysis method: differential gene screening was done using Limma R Package;the enriched differential functions and pathways were detected using the DAVID database;Gene Set Enrichment Analysis(GSEA)was used to further verify the enriched differential functions and pathways; the mutation gene manifestation was described through the maftool R package;nomogram was used to estabolish and evaluate the prognostic score system.(3)Cell line model: Human acute myeloid leukemia cell lines KASUMI-1,HL60,THP-1,U937 and MOLM14 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; The murine leukemia cell line AE-9a was presented by Chinese Academy of Sciences,Shanghai Institute of Nutrition and Health.(4)Molecular biology experiment: qPCR and WB were used to verify molecular expression;Colony formation experiment was used to detect the clone ability of AML cells;cck8 was used to determine cell proliferation viability;ATP kit was used to detect the content of ATP.(5)Flow cytometry experiment: Flow cytometry related experiments are used for apoptosis analysis,reactive oxygen content analysis,mitochondrial membrane potential analysis and DNA damage analysis.(6)Statistical analysis: Chi-square analysis and independent sample t test were used to investigate whether there were differences between grouping variables.Cox regression analysis was used to determine the factors that affect the survival and prognosis of patients.The Kaplan-Meier curve was used to describe the survival status of patients,and the log-rank test was performed to compare the differences in survival status between patients in different groups.The correlation analysis was completed by Pearson test.Graph Pad Prism 7 and R3.6.1 were used to complete the above related analysis.The two-tailed p value less than 0.05 was defined as the standard for the difference of statistical significance.Results:First partThrough CRISPR genome-wide screening open data,we found SLC25A1,a potential new molecular marker for acute myeloid leukemia.Through database analysis and PCR verification,we found that SLC25A1 was significantly up-regulated in AML and was related to the poor prognosis of patients.Through cell function experiments,it was found that knocking down SLC25A1 could inhibit the proliferation and cloning ability of AML cells,and promote the apoptosis of AML cells.Combined with transcriptome sequencing,metabolome and lipidome mass spectrometry,we found that SLC25A1 could affect the lipid metabolism,nucleotide metabolism and amino acid metabolism of AML cells;and down-regulation of SLC25A1 could damage mitochondrial function,resulting in increased reactive oxygen species and decreased ATP content,thereby inhibiting the survival of AML cells.CTPI2,a drug targeting SLC25A1,could also inhibit the survival of AML cells.At the same time,CTPI2 could enhance the DNA damage ability of the chemotherapy drug DNR by increasing the level of intracellular reactive oxygen species,thereby cooperating with DNR to promote apoptosis and inhibit proliferation.CTPI2 and the BCL-2 inhibitor venetoclax could also synergistically inhibit the growth of AML cells by promoting apoptosis.Finally,the fatty acid metabolism model containing the SLC25A1 gene that we constructed could better evaluate and predict the prognosis of AML patients.Second partThrough the differential gene analysis and prognosis analysis of the databases,we found that HOXB5 was up-regulated in AML patients and was related to the progression and poor prognosis of AML,even in different subgroups.Through the analysis of mutation data in TCGA,we found that AML patients with high HOXB5 expression had the unique gene mutation pattern,which was concurrent FLT3-ITD,NPM1 and DNMT3 A mutations.Through the functional enrichment analysis of multiple databases,we found that in AML patients,the high expression of HOXB5 was closely related to the differentiation process of myeloid cells and the signature of LSCs,and this regulation might play a role through the TNF/NF-?B pathway.Through the analysis of DNA methylation data in the TCGA database,we found that the expression of HOXB5 and its related HOX family genes were regulated by their own methylation levels,and then played a role in the occurrence and development of AML.Conclusion:First partSLC25A1 is a new key factor that regulates the metabolism of AML cells.Down-regulating SLC25A1 can damage mitochondrial function and inhibit the survival of AML cells.The application of drugs that target SLC25A1 and the combination with existing clinical drugs has certain significance for the treatment of AML.Second partHOXB5 is associated with the malignant development of AML and may be a treatment target and a biomarker for AML prognosis prediction.