| [Objective]To optimize the method of exosomes extraction,and to compare the separating efficiency of each method.Based on the second-generation sequencing,profiles of miRNAs in exosomes derived from peripheral blood of AD patients were analyzed by bioinformatics.We aim to explore the mechanism of miRNA-150-5p in exosomes regulating the proliferation,apoptosis,activation and inflammation of KCs by targeting HMGA2.[Methods]①Peripheral blood samples of patients with AD and healthy volunteers were collected and exosomes derived from serum were extracted by ultrahigh speed centrifugation and exoEasy Maxi kit,respectively.The separating efficiency of exosomes was verified by electron microscopy and NTA.②The miRNAs in exosomes and mRNA both derived from peripheral blood of AD patients and healthy volunteers were detected by Second generation sequencing.R 3.4.1 was used to screen miRNAs differentially expressed.The most valuable miRNA was selected for analysis in combination with literature review.Targetscan human 7.1 database was applied to predict the miRNA target genes,and the sequencing differential mRNA was involved for intersection.Cytoscape3.5.1 and its plugins ClueGO and CluePedia were used to enrich the GO function and KEGG signal pathway of genes.③Peripheral blood samples of 30 AD patients and 30 healthy volunteers were collected.The protein in exosomes was extracted and the protein concentration was determined.Exosomes of protein concentration 0μg/μl,20μg/μl,40μg/μl and 80μg/μl derived from healthy volunteers were respectively co-cultured with KC to find the optimum experimental concentration by detecting the proliferation and apoptosis of the cells.KCs were divided into three groups according to the source of exosomes:AD group,normal human group and control group.Markers of activation(K16)and inflammation(TSLP,IL-25,IL-33,CXCL1,CXCL2)were tested using q-PCR.④The level of miRNA-150-5p in exosomes derived from peripheral blood of AD patients and healthy volunteers was screened by means of q-PCR and the efficacy of miRNA-150-5p in diagnosis of AD was evaluated by ROC curve.HMGA2,related to cell proliferation,apoptosis and inflammation,was selected for further study based on the sequencing results and literature review.The expression of HMGA2 in the skin tissues of AD patients and healthy people was compared by immunohistochemistry and confocal laser.According to miRNA-150-5p transfected KC was divided into three groups:mimic group,inhibitor group and control group.The proliferation rate was measured by CCK-8 and the method of flow cytometry detecting Ki67.Annexin V-FITC/PI double staining was applied to test the apoptosis of cells in each group.WB was atopted to examine the expression of HMGA2.Q-PCR was undertaken to determine the activation(K16,SKALP),differentiation(K10,IVL,FLG,LOR),inflammation(TSLP,IL-1,IL-20,IL-24,IL-25,IL-33,TNF-α,CXCL1,CXCL2)of KCs in each group.The interaction between miRNA-150-5p and HMGA2 was detected by double luciferase report.⑤According to the transfection of miRNA-150-5p and HMGA2,KCs was divided into 6 groups:blank group(without any stimulation),idling group(with lipo only),siHMGA2 group(small interfering RNA),mimic group,inhibitor group and inhibitor+siHMGA2 group(inhibitor+small interfering RNA).Q-PCR was used to detect the difference of miRNA-150-5p and HMGA2 levels in each group to verify the transfection efficiency.The expression of Bcl-2,Bax,Caspase-3,STAT3,NF-κB,TGF-β and Smad2 were detected by q-PCR and WB.[Results]①Both methods successfully extracted exosomes-Uniform micro vesicles were observed under the transmission electron microscope,and the particle size of the extract was between 3nm and 200nm under the NTA microscope.However,the results showed that the purity of exosomes extracted by the kit method was higher than that by the ultrahigh speed centrifugation method.②A total of 19 miRNAs were differentially expressed in exosomes derived from peripheral blood of AD patients and healthy volunteers,of which 14 miRNAs were upregulated and 5 miRNAs were downregulated.The most valuable miRNA-150-5p was selected for further analysis.Database predicted that there were 351 target genes,and there were four genes,PAX5,MUC21,SOGA3 and HMGA2,in the intersection of the sequencing differential mRNA.GO annotation obtained 1339 biological process annotation information,204 cell component annotation and 130 molecular functional annotation information.The genes are mainly enriched in posttranscriptional regulation of gene expression,nuclear transport,regulation of translation,cell-substrate junction,mRNA binding and so on.KEGG analysis showed that the genes were significantly enriched in 82 pathways,including STAT3,NF-κB,TGF-β/Smad2 and apoptotic signaling pathways.③Compare with other groups,the proliferation rate of KC with 40 μg/ul exosome was significantly lower and the apoptotic rate was higher(P<0.001,P<0.001).Compared with the control group and normal human group,the expression of K16,TSLP,IL-25,IL-33,CXCL1 and CXCL2 in the AD group increased in different degrees(P<0.05).④Compared with healthy volunteers,the level of miRNA-150-5p in exosomes derived from peripheral blood of AD patients decreased significantly(P=0.0018).ROC curve showed that the area under the curve of miRNA-150-5p was 0.723(95%CI 0.584～0.863).It is showed that the positive expression of HMGA2 in the skin tissues of AD patients was higher than that of healthy people.Compared with inhibitor group and control group,there is a significantly lower expression of HMGA2 in mimic group(P<0.001);the proliferation rate decreased and the apoptosis rate increased(P<0.001,P<0.001);K16,SKALP,K10,IVL,TSLP,IL-1,IL-20,IL-24,IL-25,IL-33,TNF-α,CXCL2 reduced(P<0.05),while the expression of LOR and FLG enhanced(P<0.05).Double luciferase report indicated that HMGA2 was the downstream target gene of miRNA-150-5p(P<0.001).⑤Compared with the blank group and the idling group,the level of miRNA-150-5p in the mimic group was significantly over-expressed(P<0.05)and there is a lower expression of miRNA-150-5p in the inhibitor group and inhibitor+siHMGA2 group(P<0.05).The level of HMGA2 in the mimic group,siHMGA2 group and inhibitor+siHMGA2 group was downward regulated(P<0.05),and that in the inhibitor group was elevated(P<0.05).A decreased expression of Bcl-2 and an increased expression of Bax and Caspase-3 were found in mimic group,siHMGA2 group and inhibitor+siHMGA2 group(P<0.05).The level of Bcl-2 in inhibitor group is up-regulated,and the level of Bax and Caspase-3 downregulated(P<0.05).The expression of STAT3 in the mimic group,siHMGA2 group and the inhibitor+siHMGA2 group was lower than that in the inhibitor group(P<0.05).[Conclusion]Ultrahigh speed centrifugation and kit method can be used to extract exosomes from peripheral blood,but the latter has higher separating efficiency.miRNA-150-5p up-regulated in exosomes from peripheral blood of AD patients and promote the proliferation,activation and inflammation of KC by targeting HMGA2 through STAT3 and apoptotic signaling pathway,thus participating in the occurrence and development of AD,which may become a target of AD diagnosis and new biotherapy. |
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