| Objective:(1)Ulcerative colitis(UC)is a progressive chronic inflammatory disease of the intestinal tract,which is prone to recurrence.Based on colonic microarray data of ulcerative colitis and healthy controls,bioinformatics was used to screen differentially expressed mirnas and mrnas,and exploratory analysis was conducted on their cellular functions,enrichment signaling pathways,and possible roles in the pathological mechanism of ulcerative colitis;(2)To investigate the effects of serum exosomes on proliferation and polarization of macrophages in patients with ulcerative colitis and healthy controls;(3)To reveal the mechanism by which exosome derived mi RNAs regulate macrophage polarization,aggravate intestinal inflammatory response and damage intestinal mucosa through intercellular communication.Methods:(1)Colon tissue samples from UC and healthy controls(HC)matched for age,sex and ethnicity were collected for mi RNA and m RNA microarray analysis,and differentially expressed mi RNAs(DEMIs)and differentially expressed genes(DEGs)were screened using the limma package of R software.Gene ontology(GO)functional enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)and disease enrichment(DO)analysis were performed for DEGs.In addition,using mi RTar Base(http: //mirtarbase.cuhk.edu.cn/php/index.php),mi RDB(http: //mirdb.org/)and Target Scan(http: //www.targetscan.org/vert＿72/)databases to predict the possible target genes of DEMIs and make intersections with DEGs.Finally,selected genes related to inflammation regulation to determine the final target genes.We applied the CIBERSORT algorithm to compare the differences in immune cell abundance between UC and HC by analyzing m RNA microarray data.Correlation analysis was conducted to identify the relationship between the screened mi RNAs and m RNAs to immune cells;(2)Serum from patients with ulcerative colitis and healthy controls was collected,and serum exosomes from patients with ulcerative colitis(UC-Exo)and healthy controls(HC-Exo)were separated by exosome isolation kit.The particle size distribution and particle concentration of exosomes were observed by nano-tracer analyzer,and total protein in exosomes was extracted and detected by Western blot(WB).CD63 and CD81 protein expression levels were detected by Western blot.Serum exosomes were labeled with CM-Dil dye and macrophages were marked with DAPI,and the effect of macrophage uptake on exosomes was observed using laser confocal microscopy.The endocytosis of exosomes and macrophages was observed by laser confocal microscopy,and macrophage proliferation was detected by CCK8.The interleukin(IL)-1β and IL-10 in the cell supernatant were detected by ELISA.Flow cytometry was used to detect the M1 macrophage marker CD86 and the M2 macrophage marker CD206;(3)The differential expression of mi R-155 in the serum exosomes of the two groups of patients was detected by quantitative real-time PCR(q RT-PCR).The expression of mi R-155,SHIP1,PI3 K and AKT in macrophages was detected after co-culture of serum exosomes with macrophages in the two groups of patients,respectively.The mi R-155 inhibitor NC,mi R-155 inhibitor were transfected into macrophages.The expression of mi R-155,SHIP1,PI3 K,AKT in macrophages was detected.Serum exosomes from the two groups of patients extracted above were co-cultured with the transfected macrophages,respectively.The endocytosis between exosomes and macrophages was observed by laser confocal microscopy,and the proliferation of macrophages was detected by CCK8.The cell supernatants were assayed for interleukin(IL)-1β and IL-10 by enzyme-linked immunosorbent assay(ELISA),and the M1 macrophage marker CD86 and the M2 macrophage marker CD206 by flow cytometry.Results:(1)Four DEMIs and 1221 DEGs were screened by the criteria of P<0.05 and ∣log2FC∣>0.5.Enrichment analysis revealed that the cellular components of differentially expressed genes were mainly concentrated in intercellular junctions,intercellular bridges,and endoplasmic reticulum lumen.Biological processes were mainly involved in the regulation of hematopoiesis as well as multiple cell differentiation.Molecular functions include receptor activator binding,phosphatase activity,and cytoskeletal composition.DO enrichment analysis suggested that differential genes were mainly associated with prostate cancer,periodontal disease and rheumatic diseases.Three databases(mi RTar Base,targescan and mi RDB)were used to predict the target genes of DEMIs.441 genes were obtained and 28 possible target genes were intersected with DEGs.The final target gene was identified as SHIP1 gene by intersecting genes related to inflammation regulation and reading related literature.Analysis of the raw microarray data showed that mi R-155-5p was highly expressed in the colonic tissues of patients with ulcerative colitis and SHIP1 was lowly expressed.The cell abundance of γδ T cells(P=0.04),M0 macrophages(P=0.01)with activated mast cells(P<0.01)was significantly higher in patients with ulcerative colitis than in controls,while activated NK cells(P=0.02)showed the opposite expression pattern,as analyzed by the CIBERSORT algorithm.Correlation analysis showed that mi R-155-5p was positively correlated with macrophage infiltration(ρ=0.52P=0.038)and SHIP1 expression was negatively correlated with the proportion of macrophages(ρ=-0.64P=0.007);(2)Serum exosomes were extracted from both groups of patients using the Exo Quick exosome extraction kit,and the exosome particle size was between 30-150 nm when analyzed by nanoparticle tracking analysis(NTA)technique.Exosome marker proteins CD63 and CD81 were detected by Western blot,indicating successful extraction of exosomes.The results of laser confocal microscopy showed that macrophage nuclei were irregularly shaped and lobulated,and exosomes with red fluorescence were clustered around macrophages with blue fluorescence,suggesting that a large number of exosomes entered macrophages after co-culture with macrophages.The proliferation results of CCK8 showed that exosome-treated macrophages from patients with ulcerative colitis proliferated significantly.The ELISA results showed that UC-Exo induced high secretion of IL-1β and low secretion of IL-10 in M0 type macrophages.Flow cytometry results showed a significant increase in the M1 macrophage marker CD86 and a significant decrease in the M2 macrophage marker CD206 in the UC-Exo group;(3)q RT-PCR results showed high expression of mi R-155-5p in exosomes from patients with ulcerative colitis.After co-culture of macrophages with HC-Exo,UC-Ex0 for 24 h,the expression of mi R-155-5p in macrophages in UC-Exo group was significantly higher,the expression of SHIP1 gene was significantly lower,and the expression of p-PI3 K and p-Akt in macrophages was increased.After macrophage transfection with mi R-155-5p inhibitor,the expression of mi R-155-5p decreased,indicating successful transfection.At the same time,macrophage SHIP1 expression increased.The results of CCK8 showed no significant changes in macrophage proliferation after co-culture of mi R-155-5p inhibitor NC and mi R-155-5p inhibitor-transfected macrophages with UC-Exo,respectively.ELISA results showed that macrophage transfected with mi R-155-5p inhibitor,pro-inflammatory cytokine IL-1βcontent significantly decreased,while anti-inflammatory cytokine IL-10 secretion significantly increased.Flow cytometry results showed that M1-type macrophage marker CD86 was significantly decreased in mi R-155-5p inhibitor group,while there was no statistical difference in M2-type macrophage marker CD206.The expression of p-Akt was reduced in macrophages transfected with mi R-155-5p inhibitor.Conclusions:(1)Bymining m RNA and mi RNA microarray data,we identified candidate biomarkers that may be involved in the pathogenesis of UC as mi R-155-5p and its target molecule SHIP1.Both are associated with immune cell infiltration,especially macrophages;(2)A large number of exosomes enter macrophages after co-culture of serum exosomes with macrophages,and UC-Exo induces polarization of M0-type macrophages to M1-type macrophages;(3)UC-Exo enters macrophages by delivering mi R-155-5p,targeting and inhibiting SHIP1 expression,activating the PI3K/Akt signaling pathway,and inducing macrophage polarization toward M1-type macrophages.Inhibition of mi R-155-5p expression prevented macrophage polarization toward M1-type macrophages. |
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