| Background Burn remains a major global health problem,and the primary treatment of the burn wounds is of great importance in the early care of burn patients.Most of the epidermis shall be damaged in deep burn wounds,thus fibroblasts around the wounds are activated and start to proliferate and migrate,secreting collagen and fibronectin to form new extracellular matrix to shrink the wound and fill the defects.Fibroblasts are important functional cells in the healing process of deep burn wounds and can be stimulated and regulated by various local or circulating substances.Accumulated reports showed that transfusion of plasma from burned animals would cause adverse post-burn changes such as increased vascular permeability and systemic edema to unburned receptors.Researchers put forward the concept of "post-burn circulating factors" to describe the circulating substances developed from severe burn,which contributed to the classic postburn changes of skin tissues.Exosomes are extracellular vesicles with phospholipid bilayer in diameters between 30 to 150 nm,carrying nucleic acid,protein or lipid molecules for intercellular interactions via body fluids.Micro RNAs(mi RNAs),regular cargos of exosomes,are short singlestranded non-coding RNA molecules with about 19-25 nucleotides in length.Circulating mi RNAs generally avoid RNA enzyme-lead degradation by binding to proteins such as Ago2 or encapsulation in microvesicles such as exosomes.Exosomal mi RNAs expressed differentially under specific physiological or pathological conditions,could serve as potential biomarkers for early diagnosis and prognosis of various diseases.Hitherto,no research on plasma exosomes from burn patients at early burn stage was reported.We believe that cells activated after severe burns may release a large number of circulating exosomes,and exudate through the microvascular barriers with significantly increased permeability post-burn.The alterations of plasma exosome-derived mi RNA profiles potentially influence the conversions of burned skin tissues.Objective To portrait the expression profile of plasma exosome-derived mi RNAs and identify differentially expressed mi RNAs at the early stage following severe burns.We hope to identify the predicted target genes of the DEMs that related to the skin tissue regeneration to construct key mi RNA/m RNA regulatory network in blood-to-tissue interactions postburn.Finally,to explore the effect of identified key mi RNA/m RNA axis on proliferation,migration,cell cycle and secretion function of human dermal fibroblasts and its mechanisms.Methods This study is mainly divided into two parts.The part I focused on the sequencing of plasma exosome-derived small RNAs at early stage following severe burn.After identifying the differentially expressed mi RNAs,bioinformatics analysis was conducted by employing gene expression database to construct the key mi RNA/m RNA regulatory network in blood-tissue interactions.(1)Blood samples from severe burn patients 4-7 days post-burn and healthy volunteers were collected to extract and characterize the plasma exosomes.A Next-generation sequencing(NGS)of exosomal small RNAs presented the small RNA profiles and differentially expressed mi RNAs(DEMs).Target genes of DEMs were predicted in the mir DIP database.(2)In the Gene expression Omnibus database(GEO),GSE8056 dataset,which recorded the gene expression matrix of deep burn skins of patients 4-7 days post-burn and normal skins,was selected.According to the threshold set at FDR<0.05 and | log2FC(fold change)| ? 1,the differentially expressed genes(DEGs)were rendered.(3)By overlapping target genes of DEMs predicted in the mir DIP and the DEGs in GSE8056,shared genes were identified as the focus genes,which may contribute to the blood-tissue interaction at early stage following severe burn.(4)A protein-protein interaction(PPI)network was constructed based on the STRING database.Key genes in the PPI network were rendered according to the degree value of the nodes,and their upstream mi RNAs in DEMs were identified.Key mi RNA/m RNA axes constructed a potential blood-tissue interactions network at early stage following severe burn.The part II focused on the effects of hsa-mi R-718/CCNB1 axis on proliferation,migration,cell cycle and secretory function of human dermal fibroblasts and its mechanism.(1)Blood samples from severe burn patients 4-7 days post-burn and healthy volunteers were collected to enrich plasma exosome mi RNAs.Droplet digital PCR(dd PCR)was employed to verify the expression of hsa-mi R-718,hsa-mi R-6754-3p and hsa-mi R-4754.(2)Human dermal fibroblasts were cultured and divided to different groups according to different processes of transfection.Control group received no transfection operation;mimics group overexpressed hsa-mi R-718 levels;mimics NC group served as the negative control group of mimics group;inhibitor group inhibited the hsa-mi R-718 expressions by transfecting mi RNA inhibitors;and the inhibitor NC group served as the negative control group of the inhibitor group.(3)Changes of the biological behaviors of human dermal fibroblasts were detected.The cell viability of human dermal fibroblasts was detected by CCK8 assay.Cell migration was detected by wound healing assay.Cell apoptosis,cell cycle distribution and proliferation index of cells were detected by flow cytometry.(4)The binding site of hsa-mi R-718 and CCNB1 were predicted in Target Scan database.Dual-Luciferase reporter assay was used to detect the luciferase activities of CCNB1 3'UTR wild type + mimics,CCNB1 3'UTR wild type + mimics NC,CCNB1 3'UTR mutant type + mimics,and CCNB1 3'UTR mutant type + mimics NC groups.Real-time quantitative PCR(RT-q PCR)was used to determine the regulation of hsa-mi R-718 on CCNB1 m RNA expression.(5)Western blot was used to detect the expression of CCNB1 protein levels in human dermal fibroblasts effected by hsa-mi R-718.Expressions of Phosphoinositide 3-kinase(PI3K),total Akt protein and phosphorylated Akt(p-Akt),?-smooth muscle actin(?-SMA)and Collagen I were also detected.Results Part I.(1)Transmission electron microscopy(TEM),nano-particle tracking analysis(NTA)and Western blot(WB)confirmed the existence of plasma exosomes.NGS indicated that mapped mi Rbase mature mi RNA reads counted as 16.05% of all reads.According to the threshold set at FDR<0.05 and | log2FC(fold change)| ? 1,85 DEMs including 14 downregulated mi RNAs and 71 upregulated mi RNAs were rendered.A total of 12901 target genes of DEMs were predicted.(2)Compared to normal skins,microarray gene expressions of partial-thickness burned skins exhibited 1861 DEGs meeting the criteria of FDR < 0.05 and |log2FC(fold change)|?1.DEGs contained 831 upregulated genes and 1030 downregulated genes.(3)By overlapping target genes of DEMs predicted in the mir DIP and the DEGs in GSE8056,1058 shared genes were identified as the focus genes.GO and KEGG analyses of the focus genes suggested possible functions and pathways of m RNA alterations related to blood-to-tissue interactions postburn mediated by exosomal mi RNAs.For the GO analysis,top 10 terms were “immune receptor activity”,“extracellular matrix structural constituent”,“carbohydrate binding”,“cytokine activity”,“glycosaminoglycan binding”,“sulfur compound binding”,“cytokine receptor activity”,“cytokine binding”,“heparin binding” and “fibronectin binding”.The KEGG analysis indicated 7 pathways: “cell cycle”,“cell adhesion molecules”,“Glutathione metabolism”,“ECM-receptor interaction”,“hematopoietic cell lineage” and “JAK-STAT signaling pathway”.(4)The 9 key genes(CDK1,CCNB1,CCNA2,BUB1 B,PLK1,KIF11,AURKA,NUSAP1 and CDCA8)in the PPI network of the focus genes pointed to 16 upstream mi RNAs in DEMs,including 4 downregulated mi RNAs(hsa-mi R-6848-3p,hsa-mi R-4684-3p,hsa-mi R-4786-5p and hsa-mi R-365a-5p)and 12 upregulated mi RNAs(hsa-mi R-6751-3p,hsa-mi R-718,hsa-mi R-4754,hsa-mi R-6754-3p,hsa-mi R-4739,hsa-mi R-6739-5p,hsa-mi R-6884-3p,hsa-mi R-1224-3p,hsa-mi R-6878-3p,hsami R-6795-3p,hsa-mi R-550a-3p,and hsa-mi R-550b-3p).A key mi RNA-m RNA network in potential blood-to-tissue interactions at early burn stage was therefore constructed.Part II.(1)The expressions of hsa-mi R-718,hsa-mi R-6754-3p and hsa-mi R-4754 in plasma exosome at early stage following severe burn were significantly different from those in normal control group(all P <0.05).And the hsa-mi R-718 was selected to explore its potential effects on burn wound healing.(2)CCK8 assay showed that hsa-mi R-718 could inhibit the cell viability of human dermal fibroblasts(P <0.001).Wound healing assay showed that the migration rate of human dermal fibroblasts with overexpression of hsa-mi R-718 was significantly decreased at 12 h and 24 h time points(all P <0.0001);while inhibition of hsa-mi R-718 significantly increased cell migration rate(all P <0.0001).Flow cytometry showed that overexpression of hsa-mi R-718 induced the apoptosis of human dermal fibroblasts,resulting in a significant increase in the proportion of apoptotic cells,including the proportion of the early apoptotic cells and the late apoptotic cells(P <0.001,P <0.05 and P <0.05,respectively).Inhibition of the expression of hsa-mi R-718 significantly reduced the proportion of apoptotic cells and the proportion of late apoptotic cells(all P <0.01),but there was no significant difference in the proportion of early apoptotic cells(P >0.05).Cell cycle detection by flow cytometry showed that overexpression of hsa-mi R-718 could significantly increase the cell proportion in the G0/G1 phase(P <0.001);there was no significant difference in the proportion of cells in the S phase(P >0.05);but a significant decrease of cells in the G2/M phase(P <0.01).And the proliferation index(PI)of cells significantly decreased(P <0.001).Inhibition ofhsa-mi R-718 expression led to significant decrease of cells in the G0/G1 phase(P <0.001),but no significant change of cell proportion in the S phase(P >0.05);the proportion of cells in G2/M phase was significantly increased(P <0.01);cell proliferation index was significantly increased(P <0.0001).(3)Compared to the mimics NC group,the luciferase activity was significantly downregulated in the hsa-mi R-718 mimics + CCNB1 wild-type group(P <0.0001).And there was no significance in the change of luciferase activity in hsa-mi R-718 mimics + CCNB1 3'UTR mutant type group(P >0.05),which indicated that hsa-mi R-718 could directly target to the 3'UTR of CCNB1 m RNA.Moreover,RT-q PCR results showed that overexpression of hsa-mi R-718 significantly inhibited the expression of CCNB1 m RNA(P <0.001).(4)WB further confirmed that hsa-mi R-718 could significantly inhibit the expression level of CCNB1 proteins(P <0.001).The protein expression levels of ?-SMA and Collagen I in the mimics group were significantly lower than those in the mimics NC group(P <0.01 and P <0.0001,respectively).The protein expression levels of ?-SMA and collagen I in the inhibitor group were significantly higher than those in the inhibitor NC group(all P <0.0001),which indicated that hsa-mi R-718 could inhibit the secretion of ?-SMA and collagen I.In addition,the levels of PI3 K and p-Akt in the mimics group were significantly lower than those in the mimics NC group(P <0.001 and P <0.0001,respectively).The levels of PI3 K and p-Akt in the inhibitor group were significantly higher than those in the inhibitor NC group(all P <0.0001).There was no significant difference in total Akt protein expressions between the two groups.Conclusion(1)The mi RNA expression profiles in plasma exosomes of severe burn patients at early stage and the gene expression profiles of burned skin tissues significantly deranged,and the target genes of DEMs highly overlapped with DEGs in skin tissues.The identified mi RNA/m RNA axes potentially constructed a blood-tissue interactions network.(2)The hsa-mi R-718 was significantly overexpressed in plasma exosomes at early stage following severe burn.Hsa-mi R-718 directly targets to the 3'UTR of CCNB1 m RNA,inhibiting the expression of CCNB1.Hsa-mi R-718/CCNB1 may inhibit the activation of PI3K/Akt signaling pathway by downregulating the expressions of PI3 K and p-Akt.Furthermore,hsa-mi R-718 inhibited the cell viability,proliferation,migration and the secretion of ?-SMA and collagen I of human dermal fibroblasts,inducing the cycle arrest of cells in the G0/G1 phase and apoptosis,which would hinder the wound healing process. |
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