The Effect Of HOXB On MDS BM-MSC Function Was Investigated Based On Biogenic Analysis

Objective:Myelodysplastic syndrome(MDS)is a clonal hematopoietic disease which is difficult to diagnose.It is more common in elderly men and has a poor prognosis.However,hematopoietic stem cell transplantation is still the only effective way in clinical practice,overcoming MDS is still a big challenge for us.Accumulating data have shown,mesenchymal stem cells(MSC)in MDS exhibit abnormal characteristics that contribute to the initiation of the disease and the transformation into acute myeloid leukemia(AML).Especially as an important component of bone marrow microenvironment(BM),MSC has always been the focus of MDS research.However,it is difficult to find common targets in studies with limited sample sizes because MDS patients are highly heterogeneous and their disease course varies significantly.In this study,the genetic differences between MDS-MSC and HD-MSC were analyzed by data sets,and the key molecules were explored and verified to provide new targets for clinical targeting MSC therapy in MDS.Methods:1.Sample data from GSE140101,GSE107963,and GSE61853 through GEO database screening were analyzed,including MSC isolated from patients with various stages of MDS and HD.2.Differentially expressed genes(DGEs)of MDS and HD under different backgrounds were screened by R studio limma package.3.Search for overlapping genes by Venn diagram;In addition,KEGG/GO analysis of these genes was performed by David.4.Four key genes HOXB3,HOXB5,HOXB6 and HOXB7 were obtained by the interaction network of simulated proteins through String and Cytoscape.Correlation analysis was performed to analyze the relationship between four key genes and hot genes related to MDS.5.Meanwhile,survival curves of AML patients with HOXB3,HOXB5,HOXB6 and HOXB7 changeed were analyzed by CBio Portal.6.Bone marrow mononuclear cells were isolated by differential centrifugation,and adherent cells were selected for isolation and culture in vitro to obtain MSC;The expression of key genes was detected by q-PCR to verify results of bioinformatics;7.After the key genes of MDS-MSC were interfered by siRNA,the apoptosis index was detected by flow cytometry,the expression of hematopoietic factors such as SCF,TPO,IGF1,IGFBP2 and CXCL12 was detected by PCR,and the cell proliferation was detected by CCK-8.8.The support of MDS-MSC for HSC hematopoietic ability was evaluated by flow cytometry in CD34+HSC co-incubated with MSC and by microscopic analysis of HSC colony formation in methylcellulose culture.9.miRwalk and GEO databases were used to screen miRNA targeting key genes.Results:1.330,660 and 477 differentially expressed genes were screened from the mRNA expression matrix of GEO database: GSE61853,GSE140101 and GSE107490,respectively.The Venn diagram analysis of DEGs in the three databases showed that 62 genes appeared more than two times in the three databases.2.DGEs are highly enriched in embryonic skeletal system morphogenesis,angiogenesis,anterior and posterior pattern regulation,sequence-specific DNA binding,platelet degranulation,growth factor activity,presynaptic membrane,etc.KEGG pathways were mainly enriched in p53 signaling pathway and MAPK signaling pathway.3.PPI protein interaction analysis locked the key genes: HOXB3,HOXB5,HOXB6 and HOXB7.4.Correlation analysis showed that the key genes were related to the expression of MDS-related factors.5.The survival of AML patients with key gene abnormalities was significantly worse than that of patients with key gene normal expression.6.In vitro,the expression of HOXB3 and HOXB7 in MDS-MSC was significantly higher than that in HD-MSC.7.Compared with the Control group,siRNA interference of HOXB3 and HOXB7 could significantly alleviate cell failure and apoptosis,promote the differentiation of MDS-MSC into osteogenesis and adipogenesis,and restore the proliferation ability of MDS-MSC after silencing HOXB3,the differences were statistically significant.8.After interfering with HOXB3 and HOXB7,the mRNA levels of hematopoietic factors increased.9.Compared with the Control group,the apoptosis ability of MDS-HSC was affected and the colony formation ability was restored after co-incubation with treated MDS-MSC.10.The comprehensive analysis of miRNA differential expression in miRwalk and MDS-MSC predicted that hsa-miR-125a-3p,hsa-miR-671-3p,hsa-miR-1207-5p,hsa-miR-4433b-3p were targeted to HOXB3 and HOXB7.Conclusion:1.HOXB3/7 was abnormally highly expressed in MDS-MSC and was positively correlated with the progression of MDS;Knocking down HOXB3/7 can improve the function of MDS-MSC cells and slow down the progression of MDS disease.2.Hsa-miR-125a-3p,hsa-miR-671-3p,hsa-miR-1207-5p and hsa-miR-4433b-3p were predicted to be potential epigenetic targets of MDS.