| Part OneObjective:To construct the miRNA-mRNA regulatory network of diabetic nephropathy and explore its meaningful molecular markers and therapeutic targets.Methods:1.We downloaded the gene microarray data GSE30528 and non-coding RNA array analysis data GSE51674 from the GEO database and processed the data.2.Differential expression genes(DEGs)and differential expression miRNAs(DEmiRNAs)were determined according to the adjusted P and |log 2 FC|.3.We used the STRING online database to screen for protein-protein interaction pairs(PPIs)of protein products of differentially expressed genes.The Cytoscape software was used to constructed the PPI network and its MCODE plug-in was used to identify the major modules in the PPI network.4.David online database was used for GO and KEGG functional enrichment analysis of differential genes in modules.5.Bioinformatics prediction software miRanda and RNAhybrid were used to predict the target miRNA of DEG,and then the common miRNA was selected from the two results and DEmiRNAs.Finally,we obtained miRNA-mRNA interaction pairs and constructed miRNA-mRNA regulatory network using Cytoscape.6.Then,we selected the DEG with the highest degree and its related miRNAs from the miRNA-mRNA network.Results:1.A total of 1153 differentially expressed genes were screened out in the GSE30528 data set,and 107 differentially expressed miRNAs were screened out in the GSE51674 data set.2.A PPI network composed of 1076 DEGs and 7839 interaction pairs is constructed according to the STRING database.The top 3 modules determined by MCODE plug-in were composed of 50 down-regulated gene products and 106 up-regulated gene products.3.The biological processes of differential genes showed that these genes were involved in the regulation of inflammatory response,immune response,apoptotic process,cell proliferation,programmed cell death,MAPK cascade,ERK1\2 cascade,NF-κB transcription factor activity,extracellular matrix tissue and other processes.KEGG functional enrichment analysis of differential genes showed that these genes were involved in ECM-receptor interaction,PI3K-Akt signaling pathway,complement and coagulation cascade reaction,Ras signaling pathway,cell adhesion molecules,primary immunodeficiency and so on.4.Finally,138 DEGs in the module were targeted by 84 DEmiRNAs,and we constructed a miRNA-mRNA regulatory network according to their interactions.The degree of NLRP3 in this regulatory network was the highest,and it can be targeted by mirna-29a,whose expression was upregulated in DN renal tissue by about 3 times compared with control samples.Conclusion:By analyzing the data sets of miRNA and mRNA of diabetic nephropathy,we constructed a miRNA-mRNA regulatory network composed of many potential interactions.The degree of NLRP3 in this regulatory network was the highest,and it can be targeted by mirna-29a.Through functional enrichment analysis and literature review,it was indicated that they may play a key role in the regulation of programmed cell necrosis.Part TwoObjectives:1.To study the regulatory relationships among pyroptosis,autophagy and apoptosis in glomerular podocytes.2.To validate that miRNA-29a can directly bind to the 3’UTR region of NLRP3 in podocytes.3.To study the regulation of miRNA-29a on pyroptosis,autophagy and apoptosis in podocytes cultured with high glucose.Methods:1.Cell culture,passage,differentiation,synchronization,transfection and grouping:We purchased human glomerular podocyte line from ATCC Corporation.Podocytes were cultured in 1640 medium containing 20U/mL y-interferon and 5%FBS at 33℃.Then,podocytes were transferred to 5%FBS 1640 medium without y-interferon for differentiation at 37℃ for 10-14 days.According to different experimental purposes,the podocytes were grouped after cell synchronization.①In the study of podocyte pyroptosis,autophagy,apoptosis regulation relationship:Normal control group(5.5mM),high glucose apoptosis group(30 mM),high glucose apoptosis plus NSA group,high glucose apoptosis plus 3MA group,high glucose apoptosis plus ZVAD-FMK group.miRNA-29a-mimics and negative control mimics were transfected with liposome 2000 into podocytes.Podocytes were grouped after transfection.②In the dual luciferase reporter assay,podocytes were divided into miRNA-29a mimics and miRNA-29a negative control groups.③When miRNA-29a overexpression regulated the pyroptosis,autophagy,apoptosis,and proliferation of podocytes,podocytes were divided into normal control group,high glucose group,and miRNA-29a-mimics group2.Experiment ①:We used CCK8 kit to detect podocyte proliferation and Western blot to detect the expression of Caspase-1 in podocytes cultured with high glucose,and qRT-PCR to detect LC3-II,IL-18,IL-1β,caspase-1 and GSDMD mRNA expression,and flow cytometry to detect the apoptosis rate of podocytes.3.experiment②:In podocytes cultured with high glucose,the expression of miRNA-29a was detected by qRT-PCR and the expression of NLRP3 protein was detected by Western blot.The direct binding of miRNA-29a to NLRP3 was verified by dual luciferase assay.4.Experiment③:Under the condition of overexpression of miRNA-29a in podocytes,we used CCK8 to detect podocyte proliferation and qRT-PCR to detect the expression of LC3-Ⅱ,caspase-1,IL-18,and IL-1β mRNA in podocytes,Flow cytometry to detect cell apoptosis rate.5.SPSS 23.0 software was used for statistical analysis of the experimental data.The quantitative data satisfying the normal distribution were represented by x±s,t-test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.P<0.05 was considered statistically significant.All experiments were repeated for 3 times.According to the experimental data,graphPad Prism 7 was used for plotting.Results:1.Experiment ①:Compared with the control group,the proliferation activity of podocytes under high glucose condition group was lower at 24h,48h,72h and 96h(P<0.05).ZVAD-FMK and NSA significantly increased the proliferation activity of podocytes under high glucose condition,and decreased apoptosis(P<0.05).However,3MA inhibited the proliferation activity of podocytes under high glucose condition and increased the apoptosis of podocytes(P<0.05).Compared with the control group,the expression of caspase-1 protein in high glucose group was up-regulated(P<0.05).Compared with the high glucose group,the mRNA expression of LC3-Ⅱ in the NSA treatment group was significantly up-regulated(P<0.01),while it was significantly decreased in the 3MA treatment group(P<0.01).The mRNA expressions of IL-18,IL-1β,Caspase-1 and GSDMD in the NSA treatment group were significantly down-regulated(P<0.01).The apoptosis rate of podocytes in the high glucose group was higher than that in the control group.ZVAD-FMK and NSA treatment could significantly reduce the apoptosis rate of podocytes cultured in high glucose,while 3MA could significantly increase the apoptosis rate.2.Experiment ②:The expression of miRNA-29a in podocytes cultured with high glucose was significantly lower than that in the control group(P<0.01),while the expression of NLRP3 was significantly higher than that in the control group(P<0.05).Compared with the miRNA-29a negative control group,the luciferase activity of the NLRP3 wild-type vector and miRNA-29a mimics co-transfection group was significantly decreased(P<0.01),while the luciferase activity of the NLRP3 mutant vector and miRNA-29a mimics co-transfected group did not change significantly.The mRNA expression of NLRP3 was significantly lower in the miRNA-29A mimics group than in the negative control group(P<0.01).These results suggest that miRNA-29a can directly inhibit the expression of NLRP3 in podocytes.3.Experiment③:miRNA-29a mimics significantly increased the proliferation activity of podocytes and decreased apoptosis under high glucose condition(P<0.05).Compared with podocytes cultured in high glucose,the mRNA expression level of LC3-Ⅱ was up-regulated in miRNA-29a mimics group,while the mRNA expression of IL-18,IL-1β and Caspase-1 was down-regulated,and the apoptosis rate of podocytes was decreased.Conclusion:1.High glucose can induce podocyte apoptosis and pyroptosis.pyroptosis inhibitors can up-regulate autophagy and down-regulate apoptosis in podocytes cultured in high glucose.Autophagy inhibitors can up-regulate cell apoptosis.That is,the pyroptosis component can negatively regulate podocyte autophagy and positively regulate its apoptosis.Autophagy components can negatively regulate podocyte apoptosis.2.High glucose induced down-regulation of miRNA-29A in podocytes.MiRNA-29a can directly target NLRP3.3.miRNA-29a inhibited NLRP3 to down-regulate the pyroptosis of podocytes induced by high glucose,alleviated the inflammatory response,up-regulated autophagy and down-regulated apoptosis of podocytes,thus playing a protective role on podocytes.This provides a new strategy and experimental basis for the treatment of diabetic nephropathy.Part ThreeObjective:To study the mortality of maintenance hemodialysis inpatients and its influencing factors,to provide a basis for improving the long-term prognosis of hemodialysis patients.Methods:A single-center retrospective cohort study was used.Inpatients with ESRD who had regular hemodialysis for more than 3 months at the Hemodialysis Center of the Department of Nephrology,the First Affiliated Hospital of Kunming Medical University from January 2011 to July 2020 were enrolled.We collected demographic data,clinical general data,laboratory indicators,and echocardiographic indicators of the participants.Kaplan-Meier method was used to estimate the survival rate,Log-rank test was used to compare the differences between groups,and Cox regression was used to analyze the influencing factors of mortality in maintenance hemodialysis patients.Results:1.A total of 252 patients were enrolled in this study,of whom 160 were male(63.5%)and 82 were diabetic patients(32.5%).Their average age is 55.43±16.85 years old.The primary cause of dialysis was diabetic nephropathy(29.4%),followed by primary glomerular disease(18.3%).The median follow-up time was 21.5 months.A total of 80 patients(31.75%)died during the follow-up period.2.The age,FRT,APTT,and eGFR of the death group were higher than those of the survival group,while ALB,Scr,Kt/V,and PTH were lower than those of the survival group(P<0.05)。3.The KM survival curve showed that the 1-year,3-year,and 6-year survival rates of maintenance hemodialysis patients were 79.1%,62.2%,and 47.1%,respectively.The median survival time of the participants was 70.3 months.4.There were significant differences between age,SV,ALB,LDL-C,TIBC,FRT,TRF,APTT(grouped by median),T2DM and gender(P<0.05).5.Multivariate Cox regression analysis showed that age(HR 5.228,95%CI 1.575-14.166),SV(HR 0.208,95%CI 0.072-0.595),LDL-C(HR 0.269,95%CI 0.096-0.758),FRT(HR 4.65,95%CI 1.532-14.118)were independently related to the death of maintenance hemodialysis patients(P<0.05).Conclusions:1.The 1-year,3-year and 6-year survival rates of MHD patients in the center were 79.1%,62.2%and 47.1%,respectively.2.Age,SV,LDL-C,FRT were independently related to the prognosis of maintenance hemodialysis patients(P<0.05).Age and FRT are risk factors for the death of maintenance hemodialysis patients,while SV and LDL-C are their protective factors. |
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