| Acute myeloid leukemia(AML)is a heterogeneous hematological malignancy,a clonal malignant proliferative disease of myeloid archaeocyte in the hematopoietic system,and a common and serious hematological disease in adults.The occurrence,development,and prognosis of AML involve many genomic and epigenetic changes,and its specific factors still need further research.Molecular screening for AML to identify genes with potential prognostic value is of great significance for the refinement,stratification,and improvement of clinical treatment plans for AML.SLIT-ROBO family was originally described as axon guidance factors,which is composed of SLIT and its receptor ROBO.SLIT consists of three subtypes: SLIT1,SLIT2,and SLIT3.ROBO consists of four subtypes: ROBO1,ROBO2,ROBO3,and ROBO4.In recent years,more and more studies have shown that SLIT-ROBO plays a role in promoting or inhibiting tumor cell growth,tumor angiogenesis,tumor cell migration and tumor microenvironment.In hematological tumors,ROBO has been proven to be associated with the occurrence and development of AML.Some studies have found that AML patients with high expression of ROBO3 have a poorer overall survival rate.However,immunohistochemical studies have shown that low expression of ROBO3 is more common in AML patients compared to the control group of non-AML.Previous studies have shown that ROBO3 was only expressed in the nervous system,and research data on its expression in the blood system was very limited and conflicting.Therefore,further research and exploration are needed on the impact of ROBO3 on AML.Through the analysis of the relevant data in the Cancer Genome Atlas(TCGA),we found that ROBO3 was related to the occurrence and development of AML.The AML patients with high expression of ROBO3 had a short overall survival period,poor cytogenetic risk category and poor prognosis.We found a significant increase in the expression of ROBO3 in bone marrow samples of AML patients.Knockdown and overexpression of ROBO3 were achieved through adenovirus,further validating the effect of ROBO3 on AML cell proliferation,adhesion,and migration.ROBO3 could affect CD34 and Peroxisome Proliferator-activated Receptor Gamma(PPARG),the degree of methylation of CG02940147,and the expression of ROBO3 in AML were correlated.ROBO3 exerted effects on AML cells through the Hippo-YAP pathway.Our research suggested that ROBO3 might play an important role in AML,serving as a new candidate prognostic biomarker and a potential therapeutic target for AML.Targeting the Hippo-YAP pathway might be effective for AML patients with high ROBO3 expression.This research paper is divided into the following three parts:Part 1 The correlation study between the expression level of ROBO3 and the prognosis of AML patientsObjective: To analyze the relevant data in the TCGA database and study the expression of ROBO3 in AML patients and its impact on AML risk stratification and disease prognosis.Collect bone marrow samples from AML patients and non-leukemia patients in our center for further validation.Methods:1.The UCSC Xena(http://xena.ucsc.edu)was used to analyze the information and the ROBO3 expression profiles of patients in The Cancer Genome Atlas(TCGA)database.Nomogram plot was generated with the‘survival(version 3.3.1)’ and ‘rms(version 6.3-0)’ R packages.Differential expression analysis was performed using ‘DESeq2(version 1.36.0)’ R packages,and genes with p.adj< 0.05 and |log2(fold change)| > 1 were identified as DEGs.Functional analysis combining expression data were performed with ‘cluster Profiler(version 4.4.4)’ and ‘GOplot(version 1.0.2)’ R package.2.Collect mononuclear cells from the bone marrow of 24 AML patients in our center as the experimental group,while collecting mononuclear cells from the bone marrow of 24 non-leukemia patients as the control group.All AML patients were diagnosed by bone marrow morphology,immunology,cytogenetics and molecular biology.Detect the expression of ROBO3 in AML patients and non-leukemia patients using RT-q PCR method.Results:1.Bioinformatics analysis showed that ROBO3 is associated with the occurrence and development of AML,and high expression of ROBO3 in AML patients is associated with poor prognosis.The results of bioinformatics analysis showed that the cytogenetic risk categories and prognosis of AML patients with high ROBO3 expression were poor.Compared with gender,cytogenetic risk category,percentage of bone marrow primitive cells(BM%),percentage of percentage of peripheral blood primitive cells(PM%)and other factors,ROBO3 expression level had a more important impact on the prognosis of AML patients.In the methylation site of ROBO3,we found a significant correlation between the methylation level of cg02940147 and the expression of ROBO3.In addition,the methylation level of this locus was significantly reduced in AML patients with low cytogenetic risk.2.The expression of ROBO3 is elevated in the bone marrow of AML patients.Compared with non-leukemia patients,the expression of ROBO3 in the bone marrow of AML patients was significantly increased.Summary:The high expression of ROBO3 is associated with the occurrence and prognosis of AML.Part 2 The effect of ROBO3 expression on AML cell proliferation,adhesion,and migrationObjective: To achieve knockdown and overexpression of ROBO3 in human acute myeloid leukemia cell line KG-1 through adenovirus vector transfection,observe the proliferation,adhesion,and migration of KG-1 cells,and understand the effect of ROBO3 on the proliferation,adhesion,and migration of AML cells in vitro.Methods:Using adenovirus as a vector to transfect KG-1 cells,achieving overexpression of ROBO3 in KG-1 cells.Detect the proliferation of KG-1 cells after overexpression of ROBO3 using MTS method.Detect the adhesion of KG-1 cells overexpressed with ROBO3.Detect the migration of KG-1 cells after overexpression of ROBO3 using Transwell’s method.Using adenovirus transfection to construct KG-1 cells with knockdown ROBO3 expression,observe the changes in the above indicators.Results:1.The expression of ROBO3 affects the proliferation of AML cells.Knockdown and overexpression of ROBO3 in KG-1 cells were achieved through adenovirus vector transfection.The transfection effect was confirmed to be effective after 48 hours transfection.Detect the proliferation of KG-1 cells after knockdown and overexpression of ROBO3 using MTS method.Inoculate cells into 96 well plates and record the activity of KG-1 cells at 48,72,and 96 hours after transfection.Subsequently,we found that knocking down ROBO3 significantly reduced the activity and proliferation rate of KG-1 cells,while overexpression of ROBO3 significantly increased the activity and proliferation rate of KG-1 cells.2.The expression of ROBO3 affects the adhesion and migration of AML cells.Through experimental observation of the adhesion of KG-1 cells with ROBO3 knockdown or overexpression,it was found that the cell adhesion to fibrinogen significantly decreased after ROBO3 knockdown,while the cell adhesion to fibrinogen increased after ROBO3 overexpression.We studied the migration of KG-1 cells with ROBO3 knockdown or overexpression by transwell experiments.Research had found that the migration of KG-1 cells was weakened after knocking down ROBO3,while the migration of KG-1 cells was enhanced after overexpression of ROBO3.Summary:The expression of ROBO3 in AML cells affects the proliferation,adhesion,and migration of AML cells.The high expression of ROBO3 plays a promoting role,while the low expression of ROBO3 plays an inhibitory role.Considering that the adhesion of leukemia cells is an important component of their extravascular migration.The high expression of ROBO3 may promote the extravascular migration of AML cells and increase their ability to infiltrate the extracellular matrix.Part 3 Related Studies on the Molecular Mechanisms of ROBO3 on AML CellsObjective: To analyze the relevant data in the TCGA database,study the differential expression of genes in AML patients with high and low ROBO3 expression,and screen out valuable expressions for research.Using adenovirus transfection to interfere with the expression of ROBO3 in KG-1 cells,observe the expression of CD34 and PPARG.Using Hippo-YAP pathway inhibitors K-975 and Verteporfin to act on KG-1 cells with overexpression and knockdown of ROBO3,observe changes in cell activity and migration,and explore the molecular mechanism of ROBO3 in AML cells.Methods:1.Compare the gene expression of AML patients with low ROBO3 expression and AML patients with high ROBO3 expression in the TCGA database.2.Knockdown and overexpression of ROBO3 in KG-1 cells were achieved through adenovirus vector transfection.Detection of CD34 and peroxisome proliferator activated receptors in KG-1 cells overexpressed with ROBO3 by RT-q PCR and Western Blot methods(Peroxisome Proliferator activated Receiver Gamma,PPARG)expression.Meanwhile,the expression of CD34 and PPARG in KG-1 cells with knockdown of ROBO3 was detected by RT-q PCR and Western Blot methods.3.Knockdown and overexpression of ROBO3 in KG-1 cells were achieved through adenovirus vector transfection.The expression of YES associated protein 1(YAP1)in KG-1 cells overexpressed with ROBO3 was detected using RT-q PCR and Western Blot methods.Meanwhile,the expression of YAP1 inKG-1 cells with ROBO3 knockdown was detected by RT-q PCR and Western Blot methods.KG-1 cells overexpressing and knocking down ROBO3 were treated with Hippo-YAP pathway inhibitor K-975 to observe cell activity and migration.Using Hippo-YAP pathway inhibitor Verteporfin to treat KG-1 cells with overexpression and knockdown of ROBO3,the sensitivity of KG-1 cells with overexpression and knockdown of ROBO3 to Verteporfin was observed.Results:1.ROBO3 promotes the expression of CD34 in AML cells and inhibits the expression of PPARG.We compared the gene expression between AML patients with low ROBO3 expression and AML patients with high ROBO3 expression in the TCGA database.AML patients with high expression of ROBO3 had a total of2965 differentially expressed genes,of which 2073 genes were upregulated and892 genes were downregulated.Combined with gene functional enrichment analysis,multiple terms showed significant functional enrichment.The results showed that AML patients with high expression of ROBO3 had higher expression of CD34 and lower expression of PPARG.Overexpression and knockdown of ROBO3 in KG-1 cells were achieved through adenovirus vector transfection.Detect the expression of CD34 and PPARG in KG-1 cells overexpressed with ROBO3 by RT-q PCR and Western Blot methods.Meanwhile,the expression of CD34 and PPARG in knockdown KG-1 cells of ROBO3 was detected by RT-q PCR and Western Blot methods.Research has found that overexpression of ROBO3 increased the expression of CD34 in KG-1 cells and inhibited the expression of PPARG in KG-1 cells.However,knockdown of ROBO3 reduced the expression of CD34 in KG-1 cells and increased the expression of PPARG in KG-1 cells.2.The biological function of ROBO3 in AML cells may be achieved through the Hippo-YAP signaling pathway.Western blot method was used to detect the expression of YAP1 in KG-1cells overexpressed and knockdown with ROBO3.We found that the expression of YAP1 was downregulated in KG-1 cells downregulated by ROBO3 knockdown,while upregulated in KG-1 cells overexpressing ROBO3.K-975 and Vitapofen are the inhibitors of the Hippo-YAP pathway.We found that after treatment with K-975,the proliferation and migration of KG-1 cells overexpressing ROBO3 were reduced.We found that overexpression of ROBO3 can also increase the sensitivity of KG-1 cells to vitapofen.ROBO3 knockout also moderately reduced the sensitivity of KG-1 cells to K-975 and Vitapofen,although there was no statistical difference in the results,we could still observe the above trend.Summary:The high expression of ROBO3 affects the expression of CD34 and PPARG in AML,resulting in higher expression of CD34 and lower expression of PPARG.ROBO3 may regulate cellular function through the Hippo-YAP pathway.Hippo-YAP pathway inhibitors may have better therapeutic effects on patients with high ROBO3 expression.Conclusions:1.The high expression of ROBO3 in AML patients is related to the poor cytogenetic risk category and poor prognosis of AML.The methylation level of cg02940147 is significantly correlated with the expression of ROBO3.2.The high expression of ROBO3 may promote the proliferation,adhesion,and migration of AML cells,promote their growth,promote their migration to the outside of blood vessels,and increase their ability to infiltrate the extracellular matrix.3.High expression of ROBO3 promotes higher expression of CD34 and lower expression of PPARG in AML.4.ROBO3 may regulate cellular function through the Hippo-YAP pathway.Hippo-YAP pathway inhibitors may have better therapeutic effects on patients with high ROBO3 expression. |
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