The Analysis Of Microrna Differential Expression In Peripheral Blood Of Patients With Or Without Oral CGVHD

Background: Oral chronic graft-versus-host-disease(Oral cGVHD)is a common complication after allogeneic hematopoietic stem cell transplantation(allo-HSCT)and has a strong impact on the quality of patient's life after transplantation.But its pathogenesis is still unclear.Micro RNA(mi RNA)mediates the production,proliferation and development of immune cell and immune response through regulating the expression of target genes and can be taken as a biological marker of disease initiation and development.Objective: To preliminarily analyze the possible regulatory role of mi RNA in the occurrence and development of oral cGVHD,through detection and bioinformatics analysis of the differential expression of mi RNA in the peripheral blood of allo-HSCT patients with oral cGVHD and those without oral cGVHD.Methods: 1.The medical history of 94 patients with HLA homozygous alloHSCT was retrospectively analyzed to explore the clinical characteristics of oral cGVHD and the affecting factors of the occurrence of oral cGVHD,including gender of patients,use of ATG,female/male donor-to-recipient combination,graft type,number of mononuclear cells,and other clinical indicators.The inclusion criteria of patients without oral cGVHD were further determined.2.Peripheral venous blood was taken from 8 ca Tses of oral cGVHD,8 cases of oral cGVHD without oral cGVHD,and 8 cases of healthy control group to construct the mi RNAs library.Illumina Hiseq 2500 platform was used for sequencing and analyzing the differential expression of mi RNAs.Through Go term and KEGG bioinformatics analysis,the signal pathway related to the occurrence of oral cGVHD was explored.3.The expressions of mi RNA-505-5p and mi RNA-769-5p in the peripheral venous blood of 15 cases of oral cGVHD,15 cases of non-oral cGVHD and 15 cases of healthy control were detected by quantitative real-time PCR,and their target genes and signal pathways were analyzed by Go term and KEGG.4.q RT-PCR was used to detect the expression level of Smad2 in peripheral blood mononuclear cells(PBMC)of 15 oral cGVHD and 15 non-oral cGVHD,and enzyme-linked immunosorbent assay(ELISA)was used to detect the level of Smad2 protein in plasma of each group.Results: 1.Retrospective analysis of 94 patients with HLA homozygous allogeneic stem cell transplantation found that the prevalence of oral cGVHD was about 46.8%.Patients who did not develop oral cGVHD within 300 days after transplantation were less likely to develop oral cGVHD later.Oral cGVHD is characterized by various features.In addition to the characteristic reticular white striated lesions,dry mouth and ulcer/erosion are the most common,with an incidence of 63.63% and 50%,respectively.The oral characterization score of patients in the oral cGVHD group was 3.81±2.06,and the VAS score was 3.31±2.13.There was a significant positively correlation between scores of manifestation and VAS.It has not been found that gender of patients,use of ATG,female/male donor-to-recipient combination,graft type,number of mononuclear cells,and other clinical indicators had impacts on the occurrence of oral cGVHD.2.The comparison between oral cGVHD group and non-oral cGVHD group showed that 549 mi RNAs were expressed in both groups and 72 mi RNAs were significantly different expression,including 32 up-regulated mi RNAs and 40 down-regulated mi RNAs.The comparison between the oral cGVHD group and the healthy control group showed that 547 mi RNAs were expressed in both groups and 327 mi RNAs were significantly different expression,including up expression of 164 and down expression of 163.The comparison between the non-oral cGVHD group and the healthy control group showed that 546 mi RNAs were expressed in both groups and 245 mi RNAs were significantly different expression,including 160 up-regulated mi RNAs and 85 down-regulated mi RNAs.Comparison of the three groups showed that 518 mi RNAs were expressed in all three groups and 191 mi RNAs were significantly different expression.mi RNAs target genes mainly focused on transcription in biochemical processes,membrane proteins in cell components and protein binding in molecular functions.The differentially expressed genes mainly focus on the cancer-related pathway and regulate the Phosphatidylinositol 3-protein kinase B(PI3K-Akt)pathway.3.The expressions of mi RNA-769-5p and mi RNA-505-5p in peripheral blood circulation were the highest in non-oral cGVHD patients,followed by oral cGVHD patients and the lowest in healthy controls.ROC analysis of mi RNA-769-5p and mi RNA-505-5p was of certain value in the diagnosis of oral cGVHD.Bioinformatics analysis shows that the occurrence of oral cGVHD may be related to the PI3K-Akt signal pathway and TGF-?/Smad signal pathway.4.The levels of Smad2 m RNA in PBMC and Smad2 protein in plasma were higher in the oral cGVHD group than those in the non-oral cGVHD group.There was a negative correlation between Smad2 m RNA and mi RNA-769-5p.Conclusion: 1.Oral cGVHD is a common complication after allo-hsct,often occurring within 300 days after transplantation.Oral cGVHD is characterized by various manifestations.In addition to the characteristic reticular white striated lesions,dry mouth and ulcers/erosions are the most common manifestations of oral cGVHD.It has not been found that gender of patients,use of ATG,female/male donor-to-recipient combination,graft type,number of mononuclear cells,and other clinical indicators have an impact on the occurrence of oral cGVHD.2.Differential expression of mi RNA may be involved in the occurrence of oral cGVHD through influencing PI3K-Akt,TGF-?/Smad s and other signaling pathways.mi RNA-769-5p and mi RNA-505-5p are of certain value in the diagnosis of oral cGVHD.3.High expression of Smad2 in peripheral circulating blood may be associated with the occurrence oral cGVHD.