Differential Expression Analyze Of MRNA In Peripheral Blood Of High Altitude Polycythemia Patients In Tibet

Objective: As a chronic mountain sickness(CMS)with the highest incidence and the greatest harm,the pathogenesis of high altitude polycythemia(HAPC)is still not fully understood.Understanding the differential expression of related genes is helpful to understand the pathogenesis of HAPC.Therefore,this study explores the influence of gene expression on the pathogenesis of HAPC through transcriptome sequencing analysis and in vitro experimental verification of HAPC patients in Tibet.Methods: A total of 37 HAPC patients and 42 healthy subjects who met Qinghai standards in the plateau area were selected.Peripheral venous blood samples were collected for RNA-seq on the Illumina Hi Seq platform.The resulting sequencing data were analyzed by bioinformatics analysis,phenotypic association analysis and weighted correlation network analysis.Through the construction of HAPC cell model in vitro,the results of the analysis were verified at the transcriptional level.Results:1.The were significant differences in multiple clinical indicators including RBC(P < 0.0001)and HGB(P < 0.0001)existed between HAPC patients and the control.According to the screening criteria of | log2 Fold Change | > 1 and P＿adj < 0.05 in RNA-seq data,550 genes were identified to be significantly differentially expressed.2.GO functional enrichment and KEGG pathway enrichment analysis showed that the up-regulated genes were mainly enriched in processes such as erythrocyte differentiation and development and homeostasis of number of cells,while the down-regulated genes were mainly enriched in categories such as immunoglobulin production and its mediated immune response,classical pathway of complement activation and other biological processes.3.The coupling analysis of differentially expressed gene(DEG)and pathological phenotypes revealed that 91 DEGs were in close correlation with in the phenotype of red blood cell volume distribution(width-CV and width—SD),they were all up-regulated in HAPC and involved in the process of erythrocyte metabolism.4.Combined with the functional annotation of DEGs and literature survey,we found that the expression of several potential genes might be responsible for sickness and pathogenesis of HAPC,namely FECH,CA1,GLRX5,EPB42,SPTB and SPTA1.5.Besides,cell type deconvolution analysis result suggested that the changes in the number of some immune cell types was decreased significantly in HAPC patients than that in the healthy control people,implying the autoimmune level of HAPC patients was affected,and there were some differences among different individuals.6.K562 cells were used as tool cells and CD235 a as a sign to verify the success of the cell model.Finally,40 μM hemin was added for hypoxia treatment for 24 hours as the condition for the construction of the cell model.7.The qRT-PCR verification showed that the m RNA expression levels of FECH and EPB42 genes in the HAPC model group(L40)were significantly higher than the normoxic control group(N0)(P < 0.0001,< 0.0001),which was consistent with the results of subject analysis.Compared with the hypoxia control group(L0),the m RNA expression levels of FECH,CA1,SPTA1 and EPB42 genes in HAPC model group were significantly increased(P < 0.0001),while the m RNA expression levels of GLRX5 genes were significantly decreased(P < 0.0001).Compared with normoxic stimulation group(N40),EPB42 m RNA expression levels in HAPC model group were significantly increased(P <0.0001),while FECH,SPTB,CA1,GLRX5 and SPTA1 m RNA expression levels were significantly decreased(P < 0.01,< 0.0001,< 0.0001,< 0.001,< 0.0001).Conclusion: Compared with the control group,HAPC patients showed significantly higher expression in pathological phenotypes related to the volume distribution width of red blood cells(RDW-CV and RDW-SD),and their individual immune level decreased to a certain extent,which may be related to the occurrence and development of HAPC.The differential expression of FECH and EPB42 genes is likely to be an important factor causing the pathogenesis of HAPC.Our findings provide a new data base for further elucidation of the pathogenesis of HAPC.In the future,we will continue to evaluate the candidate genes identified in this study and further study the developmental mechanism of HAPC.