Differential Expression Of Exosomal MiRNAs In Apheresis Platelet Concentrates During Storage And Preliminary Study About The Effect Of Differential MiRNAs On Human Monocytes

BackgroundIt is well known that platelet transfusion is an essential clinical treatment used to restore and maintain hemostasis and clotting function.However,even such mature procedure still involves safety issues and concerns.Adverse transfusion reactions(ATRs)may occur in patients who have received prolonged platelet concentrates.Statistically,platelet concentrates have a high risk of immune-related ATRs,such as transfusion-related acute lung injury(TRALI),dyspnea,and anaphylaxis reactions.Platelet storage lesions occur during banking,which lead to latelet activation,activation of coagulation factors and accumulation of bioactive substances such as sCD40L,mtDNA and exosomes.In the course of blood transfusion,the infusion of platelet suspension containing a variety of bioactive substances can promote the formation of thrombus,TRALI and other adverse events.Studies have shown that under different pathophysiological conditions,platelet-derived exosomes can transport a variety of bioactive substances such as proteins,lipids,nucleic acids to monocytes,neutrophils and other cells to promote signal exchange between cells and also mediate inflammation.Therefore,exosomes have long been considered as potential participants in transfusion-related immune responses.Monocytes are one of the important immune cells.Exosomes can promote monocytes to express intercellular adhesion molecules by transferring arachidonic acid,thus promoting the activation of monocytes.They can also promote the recruitment of monocytes to inflammatory sites by carrying CCL5,so that monocytes can undergo cell differentiation and participate in inflammatory reactions.Platelet-derived exosomes also contain a variety of non-coding RNAs,among which miRNA is the most abundant.miRNA can complement and pair with target gene 3 ’-UTR to achieve target mRNA degradation or translation inhibition.However,it has not been clear whether exosome miRNAs in platelets during storage can activate monocytes and their effects on monocyte function.Therefore,by analyzing the effects of exosomes in apheresis platelet concentrates during different preservation periods on the inflammatory function of monocytes,and analyzing the differential expression of exosomal miRNA in apheresis platelet concentrates after 1 and 5 days of storage,this study preliminarily elucidates the molecular mechanism of the effects of exosomes from apheresis platelet concentrates during storage on monocyte function from the perspective of miRNA.Objective1.Determine the inflammatory effect of exosomes from apheresis platelet concentrates during storage on human monocytes,screen the differentially expressed mRNA of human monocytes by transcriptomic detection,and determine the signaling pathways which differentially expressed mRNA involve.2.Analyze the differential expression profiling of exosomal miRNAs from apheresis platelet concentrates on day 5 versus day 1.Screen and validate the candidate up-regulated miRNAs.Bioinformatics analysis is used to predict the target genes of the differentially expressed miRNAs and signalling pathways.This study can elucidate preliminarily the molecular mechanism of pro-inflammatory effects of exosomes from apheresis platelet concentrates with different storage time on human monocytes.Methods1.Exosome extraction and identification from apheresis platelet concentrates of different storage time1.1 Fresh apheresis platelet concentrates were collected and divided into two bags with sterile tubing welder.After storing for 1 day(D1)and 5 days(D5),20mL platelet suspension were taken from each sample to extract exosomes by differential ultracentrifugation.The exosomes were suspended in 200μL PBS and frozen at-80℃.1.2 Exosome morphology was detected by transmission electron microscopy,particle size range was detected by nanoflow cytometry,and exosome markers CD63 and CD81 were detected by Western blot.2.The type exosomes act on monocytes Exosomes(PKH26 labeled)and monocytes(DAPI labeled)were stained and co-cultured.Confocal microscopy was used to colocate exosomes and monocytes to determine whether exosomes could be taken up by monocytes and the type of phagocytosis.3.Effects of D1 exosomes and D5 exosomes on monocyte function3.1 Peripheral blood mononuclear cells(PBMC)were extracted from human fresh whole blood,and then monocytes from PBMC were extracted by magnetic bead method for the next experiment.3.2 The D1 exosomes,D5 exosomes and the equivalent volume of PBS were co-cultured with human monocytes for 24h,respectively.Monocyte morphology was observed under the microscope,and the cells were collected.TRIzol method was used to extract total RNA from the cells and perform reverse transcription.mRNA levels of cytokines TNF-α,IL-1β,IL-6,CXCL10,IL-4,IL-10 and markers CD68,CD80,CD163,and CD206 were detected by qPCR,and GAPDH was used as an internal reference for correction.3.3 Transcriptomic sequencing was performed on human monocytes stimulated by D1 exosomes,D5 exosomes,and equal volume of PBS.A total of 4 bags of apheresis platelet concentrates and human monocytes extracted from 4 fresh whole blood samples were used.By comparing the mRNA level changes of D1 exosom-stimulated monocytes and PBS-stimulated monocytes,and comparing the mRNA level changes of D5 exosom-stimulated monocytes and PBS-stimulated monocytes,differential expression mRNAs were screened out and bioinformatics analysis was conducted on these mRNAs,including GO analysis and KEGG analysis.4.Screened and verified the differentially expressed exosomal miRNAs from apheresis platelet concentrates,and preliminarily identify the miRNAs with regulatory effects on monocytes4.1 Four bags of apheresis platelet concentrates stored for 5 days and 1 day were used to compare the differential expression exosomal miRNA.The quantile algorithm was used to standardize the data,and the differential expression profiles of miRNA were obtained by T-test and fold change value screening.4.2 A total of 15 bags of apheresis platelet concentrates were verified by qPCR.Bioinformatics analysis was performed on differentially expressed miRNAs,including target gene prediction,GO analysis and KEGG analysis.4.3 Combined with the results of bioinformatics analysis of differential expression of mRNA and miRNA,the common signaling pathway was screened out.4.4 The miRNAs enriched in common signaling pathways and their target genes were determined,and the miRNAs with regulatory effects on monocyte function were preliminarily identified.Results1.Exosome extraction and identification from apheresis platelet concentrates Transmission electron microscopy revealed the morphological features of the cup-shaped bimembrane vesicles.Nanoflow cytometry showed that the size distribution was 30-150 nm.The exosomal surface markers CD63 and CD81 tested positive and calnexin tested negative in the Western blot procedure.2.The type exosomes act on monocytes Confocal microscopy photos indicated that intact exosomes could be taken up by monocytes and distributed surround the nucleus,suggesting that exosomes might enter monocytes via endocytosis.3.Effects of D1 exosomes and D5 exosomes on monocyte function3.1 The morphological changes of human monocytes were observed under ordinary microscope after co-culture with D1 exosomes and D5 exosomes.3.2 Fold change of transcription levels of inflammatory cytokines and surface markers were detected by qPCR.mRNA levels of TNF-α,IL-1β,IL-6,CXCL10,and CD 80 in human monocytes were elevated after stimulating by D5 exosomes compared with D1 exosome,suggesting that exosomes from apheresis platelet concentrates primarily played a pro-inflammatory role on human monocytes.3.3 A total of 118 different mRNAs were detected in human monocytes stimulated by D1 exosome and PBS(D1 vs PBS),of which 26 were up-regulated and 92 were down-regulated.The signaling pathways of differentially expressed mRNA mainly enriched included chemokine signaling pathway,NF-κB signaling pathway,p53 signaling pathway,apoptosis,IL-17 signaling pathway and TNF signaling pathway,etc.A total of 162 different mRNAs were detected in human monocytes stimulated by D5 exosome and PBS(D5 vs PBS),of which 47 were up-regulated and 115 were down-regulated.The signaling pathways of differentially expressed mRNA mainly enriched included apoptosis,MAPK signaling pathway,chemokine signaling pathway,toll-like receptor signaling pathway,IL-17 signaling pathway and TNF signaling pathway,etc.4.Screened and verified the differentially expressed exosomal miRNAs from apheresis platelet concentrates,and preliminarily identify the miRNAs with regulatory effects on monocytes4.1 Microarray results and bioinformatics analysis of differentially expressed miRNAs The microarray detected 134 miRNAs expressed differently in the two groups(D5 vs D1).Of these,57 miRNAs were upregulated and 77 were down-regulated(|fold change|>2.0,P<0.05).Target gene prediction and bioinformatics analysis were performed for all up-regulated miRNAs.The analysis results showed that the enriched genes might be involved in mRNA 3’-UTR binding,transcriptional regulation,regulation of serine/threoninase activity,nervous system development,cell proliferation,etc.The significantly enriched signaling pathways included AMPK signaling pathway,ErbB signaling pathway and Ras signaling pathway,all of which were closely related to immune regulation.4.2 qPCR validation and bioinformatics analysis of significantly differentially expressed miRNAsThirteen miRNAs detected in all samples were selected for verification.The results were presented as relative expression changes,5(miR-22-3p,miR-223-3p,miR-21-5p,miR-23a-3p,and miR-320b)of them increased more than 10-fold(P<0.001);4(let-7a-5p,miR-25-3p,miR-126-3p,and miR-320c)increased more than five-fold(P<0.001),2(miR-342-3p,miR-320d)of them increased more than 2-fold(P<0.05);2(miR-328-3p,miR-320e)of them increased more than 2-fold(P>0.05).The top3 miNRAs were miR-22-3p,miR-223-3p and miR-21-5p with average fold change of 14.56,12.98 and 11.96 respectively.Target gene prediction and bioinformatics analysis were performed on 9 miRNAs with average fold change≥ 5.GO analysis results showed that the enriched genes could participate in transcription regulation,post-transcriptional regulation,regulation of cell proliferation,and development of nervous system,etc.KEGG analysis showed that FoxO signaling pathway,ErbB signaling pathway,MAPK signaling pathway and TNF signaling pathway were significantly enriched,which were closely related to immune regulation.4.3 Preliminarily identified the miRNAs with regulatory effects on monocytesCompared with exosomes from D1 apheresis platelet concentrates,exosomes from D5 apheresis platelet concentrates made monocytes secrete more pro-inflammatory cytokines and surface marker.Compared with the transcriptome results of D5 vs PBS and the microarray results of D5 vs D1,three common signaling pathways were found including apoptosis,TNF signaling pathway and MAPK signaling pathway.After screening the target genes enriched in the three pathways and their corresponding miRNAs,as well as the expression of the target genes in the transcriptomic results,it was found that KITLG was significantly down-regulated(P<0.05),and it was speculated that miR-320b might bind to the target gene KITLG and impact the function of monocytes by participating in the MAPK signaling pathway.Conclusions1.Exosomes from apheresis platelet concentrates can be taken up by monocytes through endocytosis and play a pro-inflammatory role on monocytes.With the prolonged storage time of apheresis platelets,the pro-inflammatory role of exosomes in suspension on human monocytes is enhanced.2.The expression of exosomal miRNAs from apheresis platelet concentrates are changed with different storage time.Bioinformatics analysis shows that the target genes of 9 significantly different miRNAs(miR-22-3p,miR-223-3p,miR-21-5p,miR-23a-3p,miR-320b,let-7a-5p,miR-25-3p,miR-126-3p and miR-320c)are mainly involved in FoxO signaling pathway,ErbB signaling pathway,MAPK signaling pathway and TNF signaling pathway.It is speculated that differential expressed miRNAs may participate in immunomodulation through these signaling pathways.3.Exosomal miR-320b might bind to KITLG and participate MAPK signaling pathway,thus regulating the function of human monocytes.