| Benign prostatic hyperplasia(BPH)is a common disease in elderly men,and a large number of patients with BPH need surgical intervention.All types of BPH surgery cannot prevent the destruction of the prostatic urethral epithelium,which is covered on the urethra and acts as a barrier against the penetration of toxic substances in the urine.The process of Urothelium re-covering wounds is called re-epithelialization or Urothelium regenerating.Wound re-epithelialization can restore the normal anatomy and barrier function of urethra,prevent the occurrence of complications from the root of issue,effectively reduce postoperative complications and improve postoperative quality of life of patients.Our team earlier research found that neo-urothelial cells differentiated from residual basal cell within the prostate tissue wound is an important cell source of repairing,the neo-urothelial cells migrated and proliferated to complete the re-epithelialization process of the wound,and the polarization of macrophages was synchronized with the wound healing process of the neo-urothelial cells.But the specific effect and mechanism are not clear.In order to further study the influence and mechanism of macrophage polarization process on neo-urothelial cells,we planned to induce macrophage polarization into M1 and M2 in vitro,extract exosomes of different subtypes of macrophages,and obtain differential expression profiles of micro RNA of M1,M2 exosomes through high-throughput sequencing analysis.Further,the effect of M1,M2 exosomal micro RNA on the proliferation and migration ability of urinary epithelial cells and the specific mechanism were explored.In addition,a mouse transvesical laser vaporization prostatectomy model was established to lay a foundation for further research in the future.The results as follow:1.Establishment of a novel mouse model simulating transvesical laser vaporization prostatectomyOne day after the surgery,urothelium expressing uroplakin(UPK)was absent in the urethral wound site,and a large number of necrotic tissues were found in the wound site.There was no UPK-positive urothelium in the wound 3 days after surgery.At 5days after surgery,monolayer urothelium expressing UPK was found in the wound site,indicating that the re-epithelization of the wound had been completed.On the 7th day after surgery,there were multiple layers of urothelium with UPK expression,indicating that the repair was completed.2.Effects of M1,M2 and their exosomes on proliferation,migration and EMT of urothelial cellsWe used CD11b + /CD86 + to label M1 and CD11 b + / cd206 + to label M2.The results showed that with the passage of time,the proportion of M1 in wound macrophages gradually decreased and the proportion of M2 gradually increased.Combined with the previous research results,we believe that the polarization process of macrophages is involved in the regulation of wound re-epithelialization.Different subtypes of macrophage for urothelial cell proliferation and migration ability is different,compared with M1,M2 can promote urothelial cell proliferation and migration,we collected the exosomes of M1 and M2 respectively,and use exosome to treat urothelial cell,and found the urothelial cells can be successfully intake exosomes.Compared with M1 exosomes,M2 exosomes can promote the proliferation and migration of urothelial cells.3.Exploration of expression profile of microRNA from M1,M2 exosomesWe induced monocytes to polarize into M1 and M2,collected M1 and M2 exosomes,and used RNA Seq and q RT-PCR to measure micro RNA expression profiles in M1,M2 exosomes.A total of 108 differentially expressed micro RNAs were found,including 93 up-regulated micro RNAs and 15 down-regulated micro RNAs.We predicted the differentially expressed micro RNA target genes and obtained a total of1063 target genes.GO enrichment analysis of these target genes found that target genes were mainly enriched in cell adhesion,nervous system development,endosomal transport and other functions.KEGG enrichment analysis revealed that target genes were mainly enriched in the regulation of pluripotent stem cells,cholinergic synapses,chronic myelogenous leukemia,cancer pathway,Rap1 signaling pathway and other signaling pathways.PPI analysis showed that the top five key genes included SNX16.Finally,q RT-PCR experiments showed that the expression levels of miR-30a-5p and miR-99a-5p in M2 exosomes were significantly higher than those in M1 exosomes.4.M2 exosomal miR-30a-5p targets SNX16/EGFR to regulate post-prostatectomy re-epithelializationWe first predicted the target genes of miR-30a-5p,including 1538 target genes predicted by miRDB database,1576 target genes predicted by star Base database and2433 target genes predicted by Target Scan database.Then,the intersection of target genes in the three databases was selected to obtain 782 target genes existing in all the three databases,the target genes in the intersection were ranked using Target Scan score.The results showed that miR-30a-5p could be predicted to target 782 target genes,among which it was more likely to target genes as SNX16.We planned to determine SNX16 as the target gene of miR-30a-5p by dual-luciferase reporter assay,and the results showed that:Compared with NC miR-30a-5p mimic,luciferase level was significantly decreased after transfection with miR-30a-5p mimic and SNX16 WT,while luciferase level was not significantly changed after transfection with miR-30a-5p mimic and SNX16 MUT.All the above results suggested that SNX16 was the target gene of miR-30a-5p.Subsequently,urothelial cells were co-cultured with M1 and M2,and the effects of different subtypes of macrophages on the expression levels of SNX16 and EGFR in urothelial cells were detected by q RT-PCR.The results showed that compared with the blank group and M1 co-culture group,the expression levels of SNX16 and EGFR in urothelial cells in M2 co-culture group were significantly decreased,while the expression levels of EGFR were significantly increased.We also treated urothelial cells with M1 and M2 exosomes,and compared with M1 exosomes,the expression of SNX16 was also significantly decreased and the expression of EGFR was significantly increased in urothelial cells treated with M2 exosomes.These results suggest that M2 and M2 exosomes can reduce the expression of SNX16 in urothelial cells and promote the expression of EGFR compared with M1 and M1 exosomes.Finally,in order to investigate whether miR-30a-5p differentially expressed in M1,M2 exosomes plays a regulatory role,we transfected NC miR-30a-5p mimic,NC miR-30a-5p inhibitor,miR-30a-5p mimic and miR-30a-5p inhibitor into M2 cells and extracted exosome from each group 24 hours later.After treating urothelial cells with exosome in each group,the proliferation ability and migration ability of urinary tract epithelial cells were detected and compared.It was found that miR-30a-5p mimic group could significantly improve the proliferation and migration ability of urinary tract epithelial cells and reduce the apoptosis rate compared with blank group and inhibitor group.We further detected the expression of SNX16 and EGFR in urothelial cells of each group,and the results also showed that miR-30a-5p mimic group could reduce the expression of SNX16 and increase the expression of EGFR in urothelial cells compared with blank group and inhibitor group.In conclusion,It is feasible to construct the transvesical laser vaporization prostatectomy mouse model by ultra-micro cystoscope instrument and 200 μm thulium laser,which can provide an animal experimental basis for further study on the mechanism of wound re-epithelialization after prostatectomy,and different subtypes of macrophages might have a regulatory effect on wound repair.Compared with M1 and exosomes,M2 and exosomes can promote the proliferation,migration and EMT of urothelial cells,there are abundantly differentially expressed micro RNAs between M1 and M2 exosomes,miR-30a-5p differentially expressed in M1,M2 exosomes can indirectly affect the expression of EGFR in urothelial cells by targeting SNX16,thus playing a role in regulating the proliferation and migration of urothelial cells.The results can provide a theoretical basis for reducing post-prostatectomy wound repair complications,but also provide new ideas for promoting post-operative wound repair involving urothelial injury. |
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