BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated ...BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated and multifactorial;Therefore,sensitive and specific biomarkers are needed.Urinary exosome originate from diverse renal cells in nephron segments and partially mirror the pathological changes in the kidney.The microRNAs(miRNAs)in urinary exosome are remark-ably stable and highly tissue-specific for the kidney.METHODS Type 2 diabetic mellitus(T2DM)patients were recruited from the Second Hospital of Hebei Medical University and were divided into two groups:DM,diabetic pa-tients without albuminuria[urinary albumin to creatinine ratio(UACR)<30 mg/g]and DKD,diabetic patients with albuminuria(UACR≥30 mg/g).Healthy subjects were the normal control(NC)group.Urinary exosomal miR-145-5p,miR-27a-3p,and miR-29c-3p,were detected using real-time quantitative polymerase chain reaction.The correlation between exosomal miRNAs and the clinical in-dexes was evaluated.The diagnostic values of exosomal miR-145-5p and miR-27a-3p in DKD were determined using receiver operating characteristic(ROC)analysis.Biological functions of miR-145-5p were investigated by performing RESULTS Urinary exosomal expression of miR-145-5p and miR-27a-3p was more upregulated in the DKD group than in the DM group(miR-145-5p:4.54±1.45 vs 1.95±0.93,P<0.001;miR-27a-3p:2.33±0.79 vs 1.71±0.76,P<0.05)and the NC group(miR-145-5p:4.54±1.45 vs 1.55±0.83,P<0.001;miR-27a-3p:2.33±0.79 vs 1.10±0.51,P<0.001).The exosomal miR-145-5p and miR-27a-3p positively correlated with albuminuria and serum creatinine and negatively correlated with the estimated glomerular filtration rate.miR-27a-3p was also closely related to blood glucose,gly-cosylated hemoglobin A1c,and low-density lipoprotein cholesterol.ROC analysis revealed that miR-145-5p had a better area under the curve of 0.88[95%confidence interval(CI):0.784-0.985,P<0.0001]in diagnosing DKD than miR-27a-3p with 0.71(95%CI:0.547-0.871,P=0.0239).Bioinformatics analysis revealed that the target genes of miR-145-5p were located in the actin filament,cytoskeleton,and extracellular exosome and were involved in the pathological processes of DKD,including apoptosis,inflammation,and fibrosis.CONCLUSION Urinary exosomal miR-145-5p and miR-27a-3p may serve as novel noninvasive diagnostic biomarkers or promising therapeutic targets for DKD.展开更多
Objective:Peritoneal fibrosis(PF)is the main cause of declining efficiency and ultrafiltration failure of the peritoneum,which restricts the long-term application of peritoneal dialysis(PD).This study aimed to investi...Objective:Peritoneal fibrosis(PF)is the main cause of declining efficiency and ultrafiltration failure of the peritoneum,which restricts the long-term application of peritoneal dialysis(PD).This study aimed to investigate the therapeutic effects and mechanisms of bone marrow mesenchymal stem cells-derived exosomes(BMSC-Exos)on PF in response to PD.Methods:Small RNA sequencing analysis of BMSC-Exos was performed by second-generation sequencing.C57BL/6J mice were infused with 4.25%glucose-based peritoneal dialysis fluid(PDF)for 6 consecutive weeks to establish a PF model.A total of 36 mice were randomly divided into 6 groups:control group,1.5%PDF group,2.5%PDF group,4.25%PDF group,BMSC-Exos treatment group,and BMSC-Exos+TP53 treatment group.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was performed to measure the expression level of miR-27a-3p in BMSC-Exos and peritoneum of mice treated with different concentrations of PDF.HE and Masson staining were performed to evaluate the extent of PF.The therapeutic potential of BMSC-Exos for PF was examined through pathological examination,RT-qPCR,Western blotting,and peritoneal function analyses.Epithelial-mesenchymal transition(EMT)of HMrSV5 was induced with 4.25%PDF.Cells were divided into control group,4.25%PDF group,BMSC-Exos treatment group,and BMSC-Exos+TP53 treatment group.Cell Counting Kit-8 assay was used to measure cell viability,and transwell migration assay was used to verify the capacity of BMSC-Exos to inhibit EMT in HMrSV5 cells.Results:Small RNA sequencing analysis showed that miR-27a-3p was highly expressed in BMSC-derived exosomes compared to BMSCs.The RT-qPCR results showed that the expression of miR-27a-3p was upregulated in BMSC-Exos,but decreased in PD mice.We found that PF was glucose concentration-dependently enhanced in the peritoneum of the PD mice.Compared with the control mice,the PD mice showed high solute transport and decreased ultrafiltration volume as well as an obvious fibroproliferative response,with markedly increased peritoneal thickness and higher expression ofα-SMA,collagen-I,fibronectin,and ECM1.The mice with PD showed decreased miR-27a-3p.Peritoneal structural and functional damage was significantly attenuated after BMSC-Exos treatment,while PF and mesothelial damage were significantly ameliorated.Additionally,markers of fibrosis(α-SMA,collagen-I,fibronectin,ECM1)and profibrotic cytokines(TGF-β1,PDGF)were downregulated at the mRNA and protein levels after BMSC-Exos treatment.In HMrSV5 cells,BMSC-Exos reversed the decrease in cell viability and the increase in cell migratory capacity caused by high-glucose PDF.Western blotting and RT-qPCR analysis revealed that BMSC-Exos treatment resulted in increased expression of E-cadherin(epithelial marker)and decreased expression ofα-SMA,Snail,and vimentin(mesenchymal markers)compared to those of the 4.25%PDF-treated cells.Importantly,a dual-luciferase reporter assay showed that TP53 was a target gene of miR-27a-3p.TP53 overexpression significantly reversed the decreases in PF and EMT progression induced by BMSC-Exos.Conclusion:The present results demonstrate that BMSC-Exos showed an obvious protective effect on PD-related PF and suggest that BMSC-derived exosomal miR-27a-3p may exert its inhibitory effect on PF and EMT progression by targeting TP53.展开更多
基金Supported by the Nature Science Foundation of Hebei Province,No.H2023104011.
文摘BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated and multifactorial;Therefore,sensitive and specific biomarkers are needed.Urinary exosome originate from diverse renal cells in nephron segments and partially mirror the pathological changes in the kidney.The microRNAs(miRNAs)in urinary exosome are remark-ably stable and highly tissue-specific for the kidney.METHODS Type 2 diabetic mellitus(T2DM)patients were recruited from the Second Hospital of Hebei Medical University and were divided into two groups:DM,diabetic pa-tients without albuminuria[urinary albumin to creatinine ratio(UACR)<30 mg/g]and DKD,diabetic patients with albuminuria(UACR≥30 mg/g).Healthy subjects were the normal control(NC)group.Urinary exosomal miR-145-5p,miR-27a-3p,and miR-29c-3p,were detected using real-time quantitative polymerase chain reaction.The correlation between exosomal miRNAs and the clinical in-dexes was evaluated.The diagnostic values of exosomal miR-145-5p and miR-27a-3p in DKD were determined using receiver operating characteristic(ROC)analysis.Biological functions of miR-145-5p were investigated by performing RESULTS Urinary exosomal expression of miR-145-5p and miR-27a-3p was more upregulated in the DKD group than in the DM group(miR-145-5p:4.54±1.45 vs 1.95±0.93,P<0.001;miR-27a-3p:2.33±0.79 vs 1.71±0.76,P<0.05)and the NC group(miR-145-5p:4.54±1.45 vs 1.55±0.83,P<0.001;miR-27a-3p:2.33±0.79 vs 1.10±0.51,P<0.001).The exosomal miR-145-5p and miR-27a-3p positively correlated with albuminuria and serum creatinine and negatively correlated with the estimated glomerular filtration rate.miR-27a-3p was also closely related to blood glucose,gly-cosylated hemoglobin A1c,and low-density lipoprotein cholesterol.ROC analysis revealed that miR-145-5p had a better area under the curve of 0.88[95%confidence interval(CI):0.784-0.985,P<0.0001]in diagnosing DKD than miR-27a-3p with 0.71(95%CI:0.547-0.871,P=0.0239).Bioinformatics analysis revealed that the target genes of miR-145-5p were located in the actin filament,cytoskeleton,and extracellular exosome and were involved in the pathological processes of DKD,including apoptosis,inflammation,and fibrosis.CONCLUSION Urinary exosomal miR-145-5p and miR-27a-3p may serve as novel noninvasive diagnostic biomarkers or promising therapeutic targets for DKD.
基金supported by the Technology Development Program of Shanghai Pudong New District(No.PKJ2021-Y34)the Excellent Young Medical Talent Training Program of Pudong Health Commission of Shanghai(No.PWRq2022-18).
文摘Objective:Peritoneal fibrosis(PF)is the main cause of declining efficiency and ultrafiltration failure of the peritoneum,which restricts the long-term application of peritoneal dialysis(PD).This study aimed to investigate the therapeutic effects and mechanisms of bone marrow mesenchymal stem cells-derived exosomes(BMSC-Exos)on PF in response to PD.Methods:Small RNA sequencing analysis of BMSC-Exos was performed by second-generation sequencing.C57BL/6J mice were infused with 4.25%glucose-based peritoneal dialysis fluid(PDF)for 6 consecutive weeks to establish a PF model.A total of 36 mice were randomly divided into 6 groups:control group,1.5%PDF group,2.5%PDF group,4.25%PDF group,BMSC-Exos treatment group,and BMSC-Exos+TP53 treatment group.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was performed to measure the expression level of miR-27a-3p in BMSC-Exos and peritoneum of mice treated with different concentrations of PDF.HE and Masson staining were performed to evaluate the extent of PF.The therapeutic potential of BMSC-Exos for PF was examined through pathological examination,RT-qPCR,Western blotting,and peritoneal function analyses.Epithelial-mesenchymal transition(EMT)of HMrSV5 was induced with 4.25%PDF.Cells were divided into control group,4.25%PDF group,BMSC-Exos treatment group,and BMSC-Exos+TP53 treatment group.Cell Counting Kit-8 assay was used to measure cell viability,and transwell migration assay was used to verify the capacity of BMSC-Exos to inhibit EMT in HMrSV5 cells.Results:Small RNA sequencing analysis showed that miR-27a-3p was highly expressed in BMSC-derived exosomes compared to BMSCs.The RT-qPCR results showed that the expression of miR-27a-3p was upregulated in BMSC-Exos,but decreased in PD mice.We found that PF was glucose concentration-dependently enhanced in the peritoneum of the PD mice.Compared with the control mice,the PD mice showed high solute transport and decreased ultrafiltration volume as well as an obvious fibroproliferative response,with markedly increased peritoneal thickness and higher expression ofα-SMA,collagen-I,fibronectin,and ECM1.The mice with PD showed decreased miR-27a-3p.Peritoneal structural and functional damage was significantly attenuated after BMSC-Exos treatment,while PF and mesothelial damage were significantly ameliorated.Additionally,markers of fibrosis(α-SMA,collagen-I,fibronectin,ECM1)and profibrotic cytokines(TGF-β1,PDGF)were downregulated at the mRNA and protein levels after BMSC-Exos treatment.In HMrSV5 cells,BMSC-Exos reversed the decrease in cell viability and the increase in cell migratory capacity caused by high-glucose PDF.Western blotting and RT-qPCR analysis revealed that BMSC-Exos treatment resulted in increased expression of E-cadherin(epithelial marker)and decreased expression ofα-SMA,Snail,and vimentin(mesenchymal markers)compared to those of the 4.25%PDF-treated cells.Importantly,a dual-luciferase reporter assay showed that TP53 was a target gene of miR-27a-3p.TP53 overexpression significantly reversed the decreases in PF and EMT progression induced by BMSC-Exos.Conclusion:The present results demonstrate that BMSC-Exos showed an obvious protective effect on PD-related PF and suggest that BMSC-derived exosomal miR-27a-3p may exert its inhibitory effect on PF and EMT progression by targeting TP53.
文摘目的:探讨SPY1(Speedy A1)和p27^(kip1)(cyclin-dependent kinase inhibitor 1B,p27)在子宫内膜癌中的表达情况及其临床意义。方法:采用生物医学信息数据库分析子宫内膜癌中SPY1、p27^(kip1) mRNA表达水平;采用免疫组织化学染色法及蛋白质印迹法检测子宫内膜癌及正常子宫内膜组织中SPY1、p27^(kip1)蛋白的表达水平,并分析其表达与患者临床病理参数、激素受体、癌症基因组图谱分子分型的关系,以及两者之间的相关性。通过Kaplan-Meier法绘制生存曲线,应用Cox比例风险模型分析影响子宫内膜癌患者的预后因素。结果:SPY1在子宫内膜癌组织中的蛋白质表达水平显著高于不典型增生的子宫内膜及正常子宫内膜组织(P=0.009),p27kip1在子宫内膜癌组织中的蛋白质表达水平显著低于不典型增生的子宫内膜及正常子宫内膜组织(P=0.001);SPY1蛋白表达与肿瘤病理分级(P=0.023)、国际妇产科协会(The International Federation of Gynecology and Obstetrics,FIGO)癌分期(P=0.003),肌层浸润深度(P=0.010)及淋巴结转移(P<0.001)有关,p27kip1蛋白表达与肿瘤病理分级(P=0.001)、FIGO癌分期(P=0.001)及淋巴结转移(P<0.001)有关;SPY1与p27kip1表达呈负相关(r=−0.563,P<0.001);Kaplan-Meier预后分析表明SPY1高表达患者生存率低于低表达患者(P<0.05),p27kip1高表达患者的生存率高于低表达患者(P<0.05)。Cox单因素及多因素分析表明FIGO癌分期(P=0.023)、病理分级(P<0.001)、淋巴结转移(P<0.001)及p27kip1表达(P<0.001)可作为子宫内膜癌患者的独立预后指标。结论:子宫内膜癌组织中SPY1蛋白呈高表达,p27kip1蛋白呈低表达,且两者之间呈负相关,可用于评估子宫内膜癌的发生和发展。
