| Chronic kidney disease?chronic kidney disease;CKD?,defined as chronic structural and functional impairment of the kidney due to a variety of causes?history of renal damage>3months?,including pathological impairment of normal and abnormal renal GFR,abnormal blood or urine composition,and imaging abnormalities,or unexplained GFR decline?<60ml/min·1.73m2?for more than 3 months.Nowadays,CKD is becoming an increasingly important public health problem,and the prevalence of CKD in China is about 10.8%[1].Renal fibrosis is a common pathological feature of CKD,which is mainly manifested in the massive accumulation of extracellular matrix and renal tubular atrophy,and is a common process leading to end-stage renal disease?ESRD?[2].Then,anti-fibrotic drugs can slow or even stop the progression of CKD.Recent new evidence suggests that miRNAs targeting renal fibrosis-related genes may be a potential new target for CKD anti-fibrosis therapy.MicroRNAs?miRNAs?are endogenous non-coding small molecular rnas that bind to the 3-'-utr of target mRNA and prevent mRNA transcripts from being translated into proteins to control gene expression.As a key regulator of organogenesis,cancer and disease,MicroRNAs have attracted increasing attention[3].Studies have shown that microRNAs play an important role in the pathogenesis of various kidney diseases.Renal fibrosis is a common pathway for the progression of various chronic kidney diseases to the end stage.Recent studies have shown that some miRNAs in renal tissue play an important role in the occurrence and development of renal fibrosis,and an in-depth understanding of their relationship can provide new therapeutic targets for the treatment of clinical renal fibrosis.Chapter 1:screening and verification of microRNAs that are differentially expressed inrenal tissues of FSGS,DN and MCD patients and normal renal tissues.Objective:to screen eligible patients with FSGS,DN and MCD and to screen and verify microRNAs that are differentially expressed in renal tissues of FSGS,DN and MCD and normal renal tissues.Methods:high-throughput microRNAs chip technology was used to screen differentially expressed microRNAs from renal tissues of FSGS,DN and MCD patients and normal renal tissues.Real-time PCR and in situ hybridization were used to verify the expression and localization of some differentially expressed microRNAs.Results:?1?According to the pathological results of renal biopsy in the department of nephrology of the first affiliated hospital of sun yat-sen university,the kidney tissues of FSGS,DN and MCD patients were screened.The results of high-throughput microRNAs chip suggested that 689differentially expressed microRNAs were found in kidney tissues of FSGS and DN patients comparedwithnormalkidneytissuesandMCDdiseasecontrols.?With FC-CUT>=1.5,P-value<=0.05 as the standard?,262 microRNAs were up-regulated and 427microRNAs were down-regulated,among which microRNA that microrna-1470 the most obvious up-regulated expression was microRNA on,while microrna-4483 was the most significantly down-regulated microRNA.?2?In situ hybridization results showed that mir-1470 and mir-4483 were located in the cytoplasm of human tubular epithelial cells.?3?Began from the 8 w db/db mice,weight and blood glucose level is significantly higher compared with same weeks of db/m mice,the 16 w db/db mice of serum creatinine and 24 h urine protein quantitative was increased significantly than db/m mice.HE,Masson,PASM,PAS staining in 16 w and 24 w db/db mice was compared with same weeks of db/m mice,glomerular hypertrophy,volume increase,glomerular mesangial matrix obvious amplification,and glomerular and renal interstitial collagen deposition is more apparent.The above results showed that db/db diabetic mice had developed diabetic nephropathy at 16w,which was more obvious at24w.FSGS mice showed significantly higher serum creatinine level and 24h urine protein quantification after 8w than control mice.HE,Masson,PASM and PAS staining showed glomerular sclerosis and obvious collagen deposition in renal interstitium.?4?Compared with the kidney tissues of db/m and control mice,mir-1470 was significantly increased in the kidney tissues of db/db diabetic mice and FSGS.The expression of mir-4483 was significantly decreased in the kidney tissues of db/db diabetic mice and FSGS.In renal tissues of DN and FSGS patients,mir-1470 was up-regulated by QPCR,while mir-4483 was significantly down-regulated.In vitro,compared with TGF-?1?-?and normal glucose concentration culture?12.5mmol/L?,the expression of mir-1470 was significantly increased in TGF-?1?+??10ng/ml?and hyperglycemic stimulation?30mmol/L?HK-2 cells,while the expression of mir-4483 was significantly down-regulated.Summary:High-throughput microRNAs chip detection results showed that,compared with normal kidney,there were 689 microRNAs with differential expressions in renal tissues of FSGS?DN and MCD patients,among which mir-1470 was the most obvious up-regulated microRNA,while mir-4483 was the microRNA with the most significant down-regulated expression.In situ hybridization showed that mir-1470 and mir-4483 were located in the cytoplasm of mouse renal tubular epithelial cells.The expressions of mir-1470 and mir-4483 in renal tissue and TGF-1 and HG-stimulated human tubular epithelial cells?HK-2?in DN,FSGS patients or mouse models were consistent with the microarray.Chapter 2:The role of mir-1470 and mir-4483 in renal fibrosis of DN and FSGSObjective:To investigate the role of mir-1470 and mir-4483 in renal fibrosis of DN and FSGS.Methods:In human renal tubular epithelial cells?HK-2?interference and overexpression of miR-1470,miR-4483,the QRT-PCR detect the expression of a-SMA,collagen I,collagen III and Fibronectin mRNA expression in HK-2 cells,Western blot,Immunofluorescence methods used to detect fibrotic changes in protein expression level in HK-2 cells.Results:?1?Transfection mimic negative control compared to transfection of miR-1470,miR-4483 mimic in HK-2 cells,the expression level of miR-1470,miR-4483 is extremely incressed.In vitro overexpression of miR-1470 can make a-SMA,Collagen I,Collagen III and Fibronectin mRNA expression increased obviously in HK-2 cells.Fibronectin,a-SMA,Collagen I,Collagen III,Collagen IV,vimentin are increased in protein level.Overexpression the level of MiR-4483 in vitro can make the a-SMA,Collagen I,Collagen III and Fibronectin mRNA expression significantly reduced,while Fibronectin,a-SMA,Collagen I,Collagen III,Collagen IV,vimentin protein expression level decreased.?2?Transfection inhibitor negative control compared to transfection of miR-1470,miR-4483 inhibitor plasmid in HK-2 cell,the expression level of miR-1470,miR-4483 is extremely decressed.In vitro inhibition of miR-1470 can make the a-SMA,Collagen I,Collagen III and Fibronectin mRNA expression significantly decreased in HK-2 cells.Fibronectin,a-SMA,Collagen I,Collagen III,Collagen IV,vimentin are decreased in protein expression level.Inhibition of miR-4483 can make the a-SMA,Collagen I,Collagen III and Fibronectin mRNA expression enhanced obviously in HK-2 cells,while Fibronectin,a-SMA,Collagen I,III Collagen,Collagen IV,vimentin increased in protein expression level.Summary:miR-1470 promotes the synthesis of fibrotic proteins in HK-2 cells and is a damaging factor,while mir-4483 inhibits the synthesis of fibrotic proteins and is a protective factor.Chapter 3:study on the mechanism of miR-1470 and miR-4483 in renal fibrosis of DNand FSGSObjective:To investigate the related mechanisms of mir-1470 and mir-4483 on renal fibrosis under the stimulation of HG and TGF-?1 in vitro.Methods:Using bioinformatics methods to predict and miR-1470,miR-4483 is associated with renal fibrosis of downstream target genes,the quantitive real time polymerase chain reaction?QRT-PCR?to detect interference or overexpression of miR-1470,miR-4483 in 293T cells with double luciferase reporter gene method validation of miR-1470,miR-4483 and its downstream target gene.Meanwihle,building mutant plasmid by mutation combining site,to determine the miRNA and binding sites of downstream target genes.Results:?1?According to the prediction of the bioinformatics website,the downstream target genes that meet the screening criteria:mir-1470 has 42 downstream target genes related to renal fibrosis,while mir-4483 has 26 downstream target genes related to renal fibrosis.?2?It was verified by QPCR that the expressions of MMP14,MMP2,MMP13,MMP9 and CTGF in 293T cells were decreased after the up-regulation of mir-1470,while the expressions of MMP14,MMP2,MMP13,MMP9 and CTGF were increased after the down-regulation of mir-1470.However,the expression of TIMP decreased after the up-regulation of mir-4483 in293T cells,and increased after the down-regulation of mir-4483.?3?The double luciferase gene reporting system showed that after co-transfection with the wild-type MMP13 vector,the luciferase activity of 293T cells was decreased after transfection with mir-1470 mimic,compared with the control mimic control and negative control.After co-transfection with a wild-type TIMP carrier,the luciferase activity of 293T cells was decreased after transfection with mir-4483 mimic,compared with mimic control and negative control.However,after co-transfection with mir-1470 mimic and MMP13 mutant plasmids,mir-4483mimic and TIMP mutant plasmids,the inhibition of luciferase activity in 293T cells was not obvious.Summary:According to bioinformatics prediction,QPCR and dual luciferase gene report verified that MMP13 was a downstream target gene of mir-1470,and TIMP was one of the downstream target genes of mirna-4483.Conclusion1.In this study,the high-throughput microRNAs chip detection results showed that,compared with MCD and normal kidney,689 differentially expressed microRNAs were found in renal tissues of FSGS?DN and MCD patients,among which mir-1470 was the most obvious up-regulated expression,while mir-4483 was the most significant down-regulated expression.In situ hybridization showed that mir-1470 and mir-4483 were located in the cytoplasm of mouse renal tubular epithelial cells.The expressions of mir-1470 and mir-4483 in renal tissues of DN,FSGS patients or mouse models and human tubular epithelial cells?HK-2?stimulated by TGF-?1 and high glucose were consistent with the microarray.2.Mir-1470 promoted the synthesis of fibrotic protein in hk-2 cells,and mir-4483 inhibited the synthesis of fibrotic protein.3.Mir-1470 can bind to the downstream target gene MMP13,thereby inhibiting its expression and promoting the process of renal fibrosis.Mir-4483 can bind to the downstream target gene TIMP and inhibit the expression of TIMP,thereby inhibiting the progression of renal fibrosis. |
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