| Background and Objective:Current studies have shown that psoriasis is a chronic immune disease induced by heredity,trauma,infection,metabolic disorders,drugs,mental and psychological factors on the background of polygenic heredity.It has a high incidence,is difficult to cure and is easy to relapse.Th1/Th2 imbalance is an important pathogenesis of psoriasis,many studies have been done in the past,but with the in-depth study of Th17 cells,there is more evidence that Th17/Treg imbalance also plays a very important role in the pathogenesis of psoriasis.IL-23 stimulates the differentiation and proliferation of Th17 cells,which makes Th17 cells secrete high levels of IL-17-mediated inflammatory response and autoimmune diseases.IL-23/Th17 pathway is an inflammatory pathway found in recent studies,which is closely related to the pathogenesis of psoriasis.As negative regulatory cells,Treg cells inhibit T cells by producing cytokines such as IL-10,IL-35,TGF-β and so on.Mesenchymal stem cells(mesenchymalstemcells,MSCs)are pluripotent stem cells and a branch of stem cells,which have the ability to self-renew and differentiate into mesenchymal cell lines,including osteoblasts,chondrocytes and adipocytes.It is the most widely used type of stem cells in clinical research,which can treat a variety of immune diseases,such as multiple sclerosis and autoimmune rheumatism.Mesenchymal stem cells exert immunomodulatory functions,inhibit cell fibrosis and apoptosis,promote cell survival,differentiation and angiogenesis through direct intercellular contact and paracrine effect.Studies have shown that MSCs in healthy people and psoriasis patients can promote the proliferation of Treg lymphocytes,but compared with healthy people,the ability of MSCs in promoting the proliferation of Treg lymphocytes is significantly reduced.Meanwhile,Patients with MSCs promoted decreased expression and secretion of nuclear transcription factor FOXP3 in Treg lymphocytes and IL-10 and TGF-β in peripheral blood lymphocytes.Exosomes are extracellular vesicles secreted by cells with a diameter of 40~100nm,which can specifically express CD9,CD63 and CD81,and contain a variety of bioactive molecules,such as RNA,proteins,lipids and enzymes.It mediates cellular communication and performs a variety of biological functions between cells.mi RNA is a class of endogenous non-coding small RNAs,about 18 to 25 nucleotides long,which have been found to be abnormally expressed in various cancers and skindiseases.By binding to the 3’UTR region of target m RNA,mi RNA promotes its degradation or inhibits its translation,thus participating in the regulation of cell proliferation,differentiation,apoptosis and other biological processes.Especially under the stress of environmental factors such as inflammation and aging,the stability of immune cell development can be seriously affected.mi RNA is a key regulator of many biological processes,such as cell development and differentiation,angiogenesis,apoptosis,metabolism and signal transduction.Exosomes are rich in mi RNA,and the exosomes secreted by MSCs can carry mi RNA for substance exchange and information exchange with target cells.mi R-155-5p has been shown to play an important role in the regulation of inflammation and immune response.R.X.Hou et al,a researcher in our Department,previously demonstrated through microarray studies that skin mesenchymal stem cells(MSCs)in patients with psoriasis can induce up-regulation of mi R-155-5p expression under the action of inflammatory cytokines such as IFN-γ,TNF-α and IL-1(significantly increased in psoriasis),while the expression of target genes TAB2 and i NOS is low.It may affect the cytokine expression and immunomodulatory function of MSCs to a great extent.However,compared with normal people,whether mi R-155-5p is also highly expressed in the exosomes secreted by psoriatic MSCs,and what are the different mi RNAs in the exosomes secreted by psoriatic and normal skin MSCs?Can binding downstream target genes play a role in Th17/Treg imbalance associated with psoriasis?The purpose of this study was to elucidate the mechanism of mi RNA targeting Th17/Treg imbalance in the exosomes of psoriatic MSCs,which may provide new potential drug targets for the treatment of psoriasis.Methods:1.MSCs were isolated and cultured from the skin tissues of normal persons and patients with psoriasis.Exosomes were extracted from the supernatant of MSCs from psoriatic lesions and normal skin by differential centrifugation.The characteristic marker CD63 on exosome surface was detected by electron microscopy,particle size analysis and Western-blot.The exocrine concentration was determined by BCA method.2.Human mesenchymal stem cell exosomes were co-cultured with human T lymphocyte lines.The ratio of Th17/Treg was detected by flow cytometry,and the contents of IL-17,IL-23,IL-10 and TGF-β in cell cultures were detected by ELISA.3.The relative contents of hsa-mi R-155-5p and external ginseng cel-mi R-39 in the samples were detected by RT-q PCR.The exosomes in the supernatant of psoriatic lesions and normal skin MSCs were sequenced by mi RNA high-throughput sequencing,and the differential mi RNAs were screened.The 9 candidate mi RNAs with significant differences were verifiedby RT-PCR,and the mi RNA with the most significant difference was selected as the research target.By using gene ontology(GO)enrichment analysis,the predicted target genes of differentially expressed mirnas were divided into three independent categories,namely biological process(BP),cellular component(CC)and molecular function(MF).Using DAVIDBioinformatics Resources6.8 GO enrichment and KEGG analysis,and used to analyze PPI STRING11.5.The online tools of Target Scan,mi RDB and mi Randa were used to predict the targetgene of 29 differential mi RNA.189 potential target genes were screened.The target genes associated with psoriasis research were screened out in the Pubmed database,and the target genes associated with psoriasis research were screened out.At the same time,mi RNA and Th17/Treg were used as keywords to predict the potential target genes of target mi RNA.RT-PCR and Western-blot further screened and verified the target genes.Double luciferase reporter vector experiment was used to verify the existence of binding sites between mi RNA and target genes.4.The selected differential mi RNAs were transfected with lentivirus from normal skin derived mesenchymal stem cells.The m RNA transcription levels of mi RNA and target genes were detected by RT-PCR,and the expression levels of target genes were detected by Western Blot.The exosomes isolated from mi RNAmimics/inhibitor mesenchymal stem cells were co-cultured with human T lymphocytes.The ratio of Th17/Treg was detected by flow cytometry,and the contents of IL-17,IL-23,IL-10 and TGF-β in cell cultures were detected by ELISA.5.All analysis using SPSS software(Version20.0,Inc.,Chicago,IL,USA).The paired t-test was used to compare paired groups.One-way ANOVA and Tukey’s test were used for statistical differences between experimental data groups.P values less than 0.05 were considered as significant differences.Results:1.The relative contents of hsa-mi R-155-5p and external parameter cel-mi R-39 in the samples were detected by RT-q PCR,and the external parameters were normal,but there was no significant difference in hsa-mi R-155-5p between psoriatic and normal skin MSCs exocrine samples.2.MSCs exosomes were co-cultured with T cell lines,and the Th17/Treg ratio was detected by flow cytometry.In the psoriatic exosome co-culture group,the differentiation of T cell lines toward Th17 increased,but the differentiation toward Treg decreased.The contents of IL-17,IL-23,IL-10 and TGF-β in cell cultures were detected by ELISA.The specific molecules IL-17 and IL-23 of Th17 were significantly increased,while the specific molecules IL-10 and TGF-β of Treg were significantly decreased.3.mi R-183-5p was screened by mi RNA high-throughput sequencing and bioinformatics analysis.Redge R package was used to analyze the difference of MSCs exosome sequencing between psoriasis and normal skin.Volcanic map and heat map were drawn by Rggplot2 package to show the differential expression of 29 kinds of mi RNA in psoriasis and normal skin(|log FC|>1&PValue<0.05).Heat map and volcano map showed that 13 kinds of mi RNA were up-regulated and 16 kinds of mi RNA were down-regulated.Bioinformatics analysis results of significantly different mi RNA predicted target genes are shown in the figure respectively.KEGG pathway analysis showed that these target genes were mainly concentrated in PI3K-Akt,FOXO,MAPK and IL-17 pathways.GO BP enrichment analysis showed that target genes were mainly enriched in RNA polymerase ⅱtranscriptional regulation,negative regulation of apoptosis and gene expression,and positive regulation of apoptosis.GO CC enrichment analysis showed that the target genes were mainly enriched in the nucleus,while GO MF enrichment analysis showed that the target genes were mainly enriched in protein binding.Nine mi RNA(mi R-4488,mi R-1246,mi R-1290,mi R-150-5p,mi R-183-5p,mi R-196a-5p,mi R-199a-5p,mi R-200b-5p and mi R-203a-3p)with more than 3 times difference from the control group were selected for RT-PCR verification.Based on the results,the expression level of mi R-183-5p in psoriatic exocrine was significantly decreased.We took it as a candidate mi RNA for follow-up study.Bioinformaticsprediction determined that the target group of mi R-183-5p was SMAD4 and FOXO1.4.q RT-PCR and WB detected the expression levels of target genes SMAD4 and FOXO1 in the normal group and the psoriasis group,and FOXO1 was up-regulated with the downregulation of mi R-183-5p,indicating that mi R-183-5p has a targeted regulatory relationship with FOXO1.Dual luciferase reporter gene assay showed that firefly Luc activity of p SICHECK2-FOXO1-WTreporter gene was significantly inhibited by mi R-183-5p-mimic(P <0.05).In the psoriasis group,mi R-183-5p was down-regulated while SMAD4 was not upregulated,indicating that SMAD4 had no targeted regulatory relationship with mi R-183-5p.5.Psoriatic MSCs exosomes can down-regulate mi R-183-5p,increase the expression of FOXO1,promote the differentiation of T cell lines into Th17,and significantly increase the expression of Th17-specific factors IL-17 and IL-23,while the exosomes isolated from mesenchymal stem cells interfered by mi R-183-5p mimics did not cause significant change in the proportion of Th17/Treg.Conclusion:1.Exosomes are selective for effector molecules of transport,and this selective mechanism determines that exosomes play a fine regulatory role in mediating intercellularcommunication,signal transduction,transportation of genetic material,immune regulation and other pathophysiological processes.2.Co-culture of psoriatic MSCs exosomes and T cell lines can cause the imbalance of Th17/Treg differentiation.3.MSCs exosomes of psoriasis can increase FOXO1 expression by downregulating mi R-183-5p,and have a targeted binding relationship between mi R-183-5p and FOXO1.4.MSCs exosomes in psoriatic lesions can down-regulate mi R-183-5p and up-regulate FOXO1,promote T lymphocyte lines to differentiate into Th17 and aggravate inflammatory response.mi R-183-5p/FOXO1 pathway can not effectively promote the differentiation of T lymphocyte lines into Treg,but there are other mechanism that affect the differentiation of Treg. |
PDF Full Text Request