| Background Oral chronic graft versus host disease(OcGVHD),a common complication after allogenetic hematopoietic stem cell transplantation,is one of the factors that affect the long-term survival of patients.However,the etiology of oral cGVHD remains unclear.Non-coding micro RNA(mi RNA)can regulate the generation,proliferation,and development of many different types of immune cells by regulating the expression of relevant target genes.At present,mi RNAs has been verified as biological markers of occurrence,development and prognosis of many different types of diseases.In our previous research,the expression of mi R-769-5p was found to had significant differences in the peripheral circulation of patients between oral cGVHD(OcGVHD)and Nonoral cGVHD(NcGVHD)groups by using high-throughput sequencing.Objective To analyze the expression of mi R-769-5p,TGF-β/SMADs pathway and M2 macrophage-related genes and proteins in peripheral blood mononuclear cells(PBMCs)of different two groups between OcGVHD and NcGVHD.The expression differences were analyzed and verified by bioinformatics to preliminarily analyze the possible regulatory role of mi R-769-5p in regulating M2 macrophage activation by targeting TGF-β/SMADs pathway in the emergence and progress of OcGVHD.Methods 1.The medical history data of 46 patients who underwent alloHSCT due to hematological diseases were retrospectively analyzed,and the clinical characteristics of OcGVHD and its relationship with clinical indicators were investigated.2.Blood samples were collected from 23 OcGVHD patients and NcGVHD patients respectively,and PBMCs were isolated.The expression levels of mi R-769-5p and TGF-β/SMADs pathway-related genes were analyzed.The enrichment pathways and biological functions of mi R-769-5p target genes were analyzed by bioinformatics techniques.3.Flow cytometry(Flow Cyto Meter,FCM)and q RT-PCR were used to detect the surface marker expression of M2 macrophages in PBMCs of 23 OcGVHD patients and NcGVHD patients respectively.4.Dual-luciferase assay was used to verify whether SMAD2 was a potential target gene of mi R-769-5p.mi R-769-5p silenced/overexpressed lentivirus was used to transfect THP-1 macrophages,and mi R-769-5p silenced/overexpressed THP-1 macrophage model was constructed to verify the regulatory effect of mi R-769-5p and SMAD2.Meanwhile,both mi R-769-5p silenced and SMAD2 silenced lentivirus were constructed to double transfect THP-1 macrophages for verification.Results 1.A total of 46 patients were observed,including 31 males and15 females,varied from 3 to 58 years,with a median age of 15 years.In our study every OcGVHD patient had the characteristic reticular white stripe lesions in their oral mucosa,and in addition to these,the most common manifestations of OcGVHD were dry mouth(with an incidence of 56.52%)and erythema lesions(with an incidence of 43.48%),respectively.The of In the OcGVHD group,oral characterization score showed a value of 3.30±2.18,and VAS score showed a value of 3.91±2.33,indicating a positive correlation between them.2.The expression of mi R-769-5p in PBMCs of OcGVHD group was significantly lower than that of NcGVHD group,and the expression of TGF-β1,SMAD2 and SMAD3 were significantly higher than that of NcGVHD group.The target gene of mi R-769-5p was predicted by various databases,and SMAD2 was found to be one of the target genes.Bioinformatics analysis showed that the molecular functions of mi R-769-5p targeted genes were mainly enriched in growth factor binding and SMAD binding,and KEGG signaling pathway analysis showed that mi R-769-5p target genes were mainly concentrated in TGF-β/SMADs signaling pathway.Protein-Protein Interaction Networks(PPI)analysis revealed that SMAD2 was a hub gene.3.FCM and RT-q PCR showed that CD11b+CD163+M2 macrophages and CD163 m RNA expression level in PBMCs of OcGVHD group was significantly higher than that of NcGVHD group.4.Dual luciferase assay verified that SMAD2 was the target gene of mi R-769-5p.The expression level of mi R-769-5p in THP1 macrophages successfully constructed with mi R-769-5p silencing lentivirus was significantly lower than that in the control group,while the expression level of mi R-769-5p in THP1 macrophages successfully constructed with mi R-769-5p overexpressing lentivirus was significantly higher than that in the control group.The expression level of TGF-β1,SMAD2,SMAD3 and CD163 in mi R-769-5p silenced THP-1macrophages were significantly higher than those in the control group,while the expression level of TGF-β1,SMAD2,SMAD3 and CD163 in mi R-769-5p overexpressed THP1 macrophages were significantly lower than those in the control group.Cell functional recovery experiments in the successful construction of both mi R-769-5p silenced and SMAD2 silenced lentivirus double transfected cell models also confirmed the correlation between mi R-769-5p and target gene SMAD2.Conclusion 1.The expression of mi R-769-5p in PBMCs of OcGVHD patients was significantly lower than that of NcGVHD patients,while the expression of TGF-β/SMADs signaling pathway and M2-type macrophages related genes were significantly higher than that of NcGVHD group.SMAD2 is a potential target gene of mi R-769-5p,and the lower expression of mi R-769-5p may be involved in the occurrence of OcGVHD by activating TGF-β/SMADs signaling pathways and M2 macrophages. |
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