| BackgroundDiabetic nephropathy(DN)is the most common complication in patients with type 1 and type 2 diabetes,and is the main cause of end-stage renal disease(ESRD).About 20%-40%of diabetic patients will eventually develop DN or even ESRD.Although the diagnosis and treatment of diabetic nephropathy has made some progress in clinical practice,however,the treatment of DN is stilla heavy burden for both patients and the country.Early clinical diagnosis of DN is usually based on urinary microalbuminuria(30-300mg/day)or the ratio of urinary albumin to creatinine(>30mg/g creatinine).However,in some patients with normal,a reduction in glomerular filtration rate(GFR)has been observed,suggesting significant glomerular damage prior to albumin detection.Data based on urinary protein alone may lead to missed diagnosis of DN.Progression to DN in diabetic patients is a very complex process,which is caused by a variety of factors.In diabetic patients,metabolic disorders lead to changes in renal hemodynamics and promote glomerulosclerosis,renal tubulointerstitial inflammation and fibrosis.Hyperglycemia and renal hypertension are known causes of DN progression.Hyperglycemia activates polyol pathways,hexosamine pathways,and protein kinase C(PKC),leading to the accumulation of advanced glycation end products(AGEs)in cells,and glomerular hyperfiltration and hypertension.An important pathophysiological feature of this stage is the activation of the renin-angiotensin system(RAS)following changes in GFR,which increases with glomerular hypertrophy,leading to increased intraglomerular pressure.Activation of the RAS system leads to dilation of glomerular entering arterioles,increasing renal blood flow and maintaining the GFR feedback mechanism.In diabetic patients,hyperinsulinemia and local angiotensin-aldosterone stimulate proximal convoluted tubule sodium reabsorption,mesangial cell proliferation,and bulbar arteriole constriction,which increase glomerular pressure and tubulointerstitial inflammation,ultimately leading to renal injury.In addition,high concentrations of angiotensin-aldosterone stimulate production of reactive oxygen species(ROS),further damaging podocytes and tubular cells.In addition,dysglycemia caused by hyperglycemia can also lead to the overproduction of ROS.The activation of activated mitogen-activated protein kinase(MAPK)and transcription factor nuclear factor-KB(NF-κB)leads to the downstream activation of tumor necrosis factor-A(TNFA).Damage DNA,and produce excessive mitochondrial growth factors such as transforming growth factor-B(TGFB),inflammatory cytokines,promote cell remodeling,further aggravate fibrosis and kidney disease,and ultimately lead to kidney failure.Autophagy is a cellular protein aggregation and damaged organelles lysosome degradation by the regulation,is of great significance to the maintenance of the steady state,attracted much attention in recent years,studies have found that it play a key role in the normal and disease states,including immunity,inflammation,stress,development and aging,metabolism and neurodegenerative diseases and cancer.The autophagy of renal tubules was inhibited and accompanied by renal hypertrophy in the rat model of diabetic nephropathy.Insulin therapy or islet transplantation could restore the autophagy activity.P62/Sequestosomel(SQSTM1),a substrate of the autophagy-lysosome degradation pathway,aggregates in diabetic nephropathy mouse and rat models,suggesting impaired autophagy.In addition,p62/SQSTM1 protein has been found to accumulate in proximal tubule cells in renal biopsy samples from patients with type 2 diabetes.Suggesting that impaired autophagy also occurs in patients with type 2 diabetes.About 2%of the human genome codes for proteins,but we still know little about the vast number of non-coding RNAs that do not encode proteins.Non-coding RNAs offer new ways to understand gene expression and regulation.That is,small non-coding RNAs(less than 200 nucleotides),including microRNAs(miRNAs)and some circRNAs(circRNAs),as well as large ncRNAs such as lncRNAs,play an important role in the occurrence and development of diseases.Recent studies suggest that non-coding RNAs(ncRNAs)play an important role in DN progression and may serve as biomarkers and therapeutic targets.In this paper,the mechanism of non-coding RNA,especially circRNA,in the occurrence and development of DN was explored through sequencing,verification and animal cell experiments of diabetic nephropathy specimens.Part one Transcriptome expression profiles associated with diabetic nephropathy developmentObjectiveThe objective of this study was to identify different transcriptome expression profiles involved in the pathogenesis of diabetic nephropathy(DN)and to illustrate the diagnosticand therapeutic potential of mRNAs,long noncoding RNAs(lncRNAs),and circular RNAs(circRNAs)in DN progression.MethodsThe participants were divided into four groups:normoalbuminuria(group DM),microalbuminuria(group A2),macroalbuminuria(group A3)and healthy controls(group N).There were three individuals in each group for sequencing.Transcriptome sequencing analysis was performed on the peripheral blood of all the participantsto identify the differential expression of mRNAs,lncRNAs,and circRNAs between intervention groups and controls.The functional enrichment analysis,the short time-series expression miner(STEM)program,and the miRNA-circRNA-mRNA network were further conducted.In order to verify the reproducibility of transcriptome sequencing,10 and 30 blood samples were collected from the control and diseased groups,respectively.Thirteen candidate biomarkers were selected from differentially expressed circRNAs(circ＿0005379、circ＿0001017、circ＿0000396、circ＿0002024、circ＿0001098、circ＿0008193、circ＿0005870、circ＿0079480、circ＿0000826、circ＿0001467、circ＿0000069、circ＿0000567、circ＿0005728)and their concentrations in the blood were measured using quantitative PCR(qRT-PCR).Results1.RNA sequencing was used to detect differentially expressed RNAs in the peripheral blood of DN patients.With the P-value<0.05,549(297 up-and 252 downregulated),1217(783 up-and434 downregulated),and 948(431 upand 517 downregulated)differentially expressed mRNAs were selected in the A2 versus N,A3 versus N,and DM versus N comparisons,respectively.With thevalues of P<0.05,1259(613up-and 646 downregulated),1613(828 upand 785downregulated),and 1495(666 up-and 829 downregulated)differentially expressed lncRNAs were selected in the A2versus N,A3 versus N,and DM versus N comparisons,respectively.In addition,with a fold change>1.5 and P<0.05 as the cut-off value,12(9upand 3 downregulated),24(9 up-and 15downregulated),and 25(3 up-and 22 downregulated)differentially expressed circRNAs were identified in the A2versus N,A3 versus N,and DM versus N comparisons,respectively.2.Functional enrichment analysis showed that differentially expressed mRNAs were related to insulin secretion,insulinresistance,and inflammation,while differentially expressed lncRNAs were mainly associated with crossover junction endodeoxyribonuclease activity.In STEM analysis,a total of 481 mRNAs and 152 differential expression circRNAs showed a significant tendency.The key relationships in the miRNA-circRNA-mRNA network wereidentified,such as hsa-miR-103 a-3p-circ＿00053 79-PTEN,hsa-miR-497-5p-circ＿0002024-IGF1R and hsa-miR-1269a-circ 0000567-SOX6.In addition,qPCR showed consistent results with RNA sequencing.ConclusionsWe found that differentially expressed mRNAs,lncRNAs,and circRNAs participated in DN development.Circ＿0005379,circ＿0002024,and circ＿0000567 could be identified as potential biomarkers for DN.Part Two The expression of circ＿0005379 in diabetic mice renal tissues and its effect on the biological behavior of human renal tubular epithelial cells treated with high glucoseObjectiveTo investigate the expression of circ＿0005379 in the kidney of diabetic model mice and its effects on the proliferation,apoptosis,fibrosis and autophagy of human renal tubular epithelial cells treated with high glucose.Methods1.Db/db and db/m mice were fed to 24 weeks,and the indexes of body weight,blood glucose,creatinine,blood lipid and 24h urinary protein were detected.Pathological changes of kidney were observed by HE staining,Masson staining,basement membrane silver hexamine staining and glycogen PAS staining.Collagen Type Ⅳ(Col Ⅳ)and a-smooth muscle action(a-SMA)were detected by immunohistochemistry.HK2 cells were stimulated with high glucose(45mmol/L)for 48 h.Total RNA was extracted from tissues and cells,and the expression of circ＿0005379 was detected by qRT-PCR.2.HK-2 cells were transfected with circ＿0005379 overexpression and knockdown vector to construct circ＿0005379 overexpression and knockdown cell model.QRT-PCR was used to detect overexpression and knockdown efficiency.The cells were divided into NC group,high glucose(HG)group,HG+mock group,HG+circ＿0005379 group,HG+siRNA NC group and HG+circ＿0005379 siRNA group.3.The effects of circ＿0005379 overexpression and knockdown on the proliferation of HK-2 cells stimulated with high glucose were detected by CCK8.4.The effects of circ＿0005379 overexpression and knockdown on the apoptosis of HK-2 cells stimulated with high glucose were detected by flow cytometry.5.QRT-PCR and western blot were used to detect the effects of circ＿0005379 overexpression and knockdown on the expression levels of fibrosis and autophagy related proteins(Col Ⅳ,a-SMA,LC3-Ⅰ,LC3-Ⅱ and p62)in HK-2 cells stimulated by high glucose.Results1.Compared with db/m mice of the same age,the body weight,blood glucose,serum creatinine,triglyceride,cholesterol,and 24-hour urine protein of db/db mice at 24 weeks were significantly increased(P<0.05).The kidney tissues of db/db mice fed to 24 weeks showed typical pathological characteristics of diabetic nephropathy,such as glomerular hyperplasia,increasing mesangial matrix,thickening of basement membrane,broadening of mesangial area,and interstitial fibrosis.Immunohistochemistry showed increased expression of Col Ⅳ and a-SMA.QRT-PCR showed compared with normal mouse kidney tissue,the expression of circ＿0005379 was significantly decreased in the renal tissues of diabetic nephropathy mice(P<0.01),in addition,the expression of circ＿0005379 was also significantly decreased in HK-2 cells stimulated by high glucose(<0.01).2.After transfection of HK-2 cells with circ＿0005379 overexpressed(circ＿0005379 group)and knockdown(circ＿0005379 siRNA group)vectors,the transfection efficiency was verified by qRT-PCR.It was found that compared with Mock group,the expression level of circ＿0005379 in circ＿0005379 overexpression group was significantly increased(P<0.01).The expression level of circ＿0005379 in cirC＿0005379 siRNA group was significantly decreased(P<0.01)compared with the knockdown vector(siRNA NC).These results indicated that the HK-2 cell model of circ＿0005379 overexpression and knockdown was successfully constructed,which could be applied in subsequent experiments.3.CCK8 results showed that HK-2 cell proliferation in HG group was significantly decreased compared with NC group(P<0.01).HK-2 cell proliferation in HG+circ＿0005379 overexpression group was significantly increased compared with HG+mock group(P<0.01).Compared with HG+siRNA NC group,the cell proliferation level of HG+circ＿0005379 siRNA group was significantly decreased(P<0.01).These results indicated that circ＿0005379 could alleviate the glucotoxicity and promote the proliferation of HK-2 cells.4.Flow cytometry results showed that the apoptosis level of HK-2 cells in HG group was significantly higher than that in NC group(P<0.01),and total apoptosis levels of HK-2 cells in HG+circ＿0005379 overexpression group were significantly lower than that in HG+mock group(P<0.01).Compared with HG+siRNA NC group,the levels of total apoptosis of HK-2 cells in HG+circ＿0005379 siRNA group were significantly increased(P<0.01).These results indicated that circ＿0005379 could alleviate the glucotoxicity and significantly inhibit the apoptosis of HK-2 cells.5.Compared with db/m mice of the same age,the expression levels of fibrosis related proteins Col Ⅳ and a-SMA in kidney tissues of db/db mice at 24 weeks of age were significantly increased(P<0.01),the ratio of autophagy related protein the expression of p62 was significantly increased,LC3Ⅱ/LC3Ⅰ was significantly decreased and(P<0.01).Conclusion1.The expression of circ＿0005379 was significantly downregulated in renal tissues of diabetic nephropathy animal models and HK-2 cells stimulated by high glucose,suggesting that the reduced expression of circ＿0005379 was correlated with the incidence of diabetic nephropathy.2.In the animal model of diabetic nephropathy and HK-2 cells stimulated by high glucose,it was found that fibrosis was aggravated and autophagy was impaired.Circ＿0005379 could alleviate the high glucose toxicity,significantly promote the proliferation of HK-2 cells stimulated by high glucose and inhibit the apoptosis.It can alleviate fibrosis and enhance autophagy by affecting the expression of ColⅣ,a-SMA,LC3-LC3-Ⅱ,p62.Part Three Mechanism of circ＿0005379 regulating biological behavior of HK-2 cells bytargetingmiR-103a-3p-PTENaxisObjectiveTo clarify the relationship between circ＿0005379,miR-103a-3p and PTEN and to identify that circ＿0005379 participates in the regulation of proliferation,apoptosis,fibrosis and autophagy of HK-2.Method1.Prediction and verification of circ＿0005379 targeted miR-103a-3p(1)Bioinformatics predicted that miRNAs targeted by circ＿0005379 included miR-103a-3p and miR-486-3p.(2)After overexpression or knockdown of circ＿0005379,the expression levels of miR-103a-3p and miR-486-3p in HK-2 cells were detected.(3)The binding of circ＿0005379 and miR-103a-3p was detected by dual luciferase reporter gene assay.(4)The localization of circ＿0005379 and miR-103a-3p as determined by fluorescence in situ hybridization.(5)The binding of miR-103a-3p and circ＿0005379 was detected by RNA immunoprecipitation.(6)The expression of miR-103a-3p in HK-2 cells and peripheral blood of diabetic nephropathy patients was detected by qRT-PCR.2.Prediction and verification of miR-103a-3p target gene PTEN(1)Bioinformatics predicted that the target genes of miR-103a-3p were KLF4,PTEN,GPRC5A and TIMP3.(2)MiR-103a-3p was overexpressed or knocked down to detect the expression levels of candidate mRNAs:KLF4,PTEN,GPRC5A and TIMP3 in HK-2 cells.(3)The binding of miR-103a-3p and PTEN was detected by dual luciferase reporter gene assay.(4)The expression of PTEN in HK-2 cells and peripheral blood of patients with diabetic nephropathy was detected by qRT-PCR.3.Effects of circ 0005379 on the expression level of PTEN:circ 0005379 was overexpressed or knocked down,and the expression level of PTEN in HK-2 cells was detected by qRT-PCR.Western blot was used to detect the expression of PTEN in HK-2 cells.4.The expression levels of miR-103a-3p and PTEN genes and proteins in kidney tissues of 24-week db/db mice and HK-2 cells stimulated by high glucose were examined by qRT-PCR and Western blot.5.HK-2 cells were transfected with miR-103a-3p inhibitor,and the transfection efficiency was tested by qRT-PCR.The cells were divided into four groups:NC group,HG group,HG+miR-103a-3p inhibitor contorl group,HG+ miR103a-3p inhibitor group.The proliferation and apoptosis of HK-2 cells were detected by CCK8 method and apoptosis flow analysis,and proteins were detected by western blot.6.MiR-103a-3p mimics were transfected in the overexpressed circ＿0005379 group,and the transfection efficiency was verified by qRT-PCR.Cells were divided into 4 groups:NC group,HG group,HG+circ＿0005379+miR-103a-3p mimics control group and HG+circ＿0005379+ mir-103a-3p mimics group The expression levels of PTEN protein were detected by western blot.7.The overexpressed circ＿0005379 group was transfected with miR-103a-3p mimics and PTEN knockdown vector,and the transfection efficiency was verified by qRT-PCR.The cells were divided into four groups for RESCUE experiment:the proliferation and apoptosis of HK-2 cells in HG group,HG+circ＿0005379 group,HG+circ＿0005379+miR-103a-3p mimics group and HG+circ＿0005379+Si-PTEN group were detected by CCK8 method and apoptosis flow cytometry.Related proteins were detected by western blot.Results1.Verification of binding relationship between circ＿0005379 and miR-103a-3p(1)The intersection of miRNAs predicted by RNA-hybrid,Targetscan and miRanda software that could be combined with circ＿0005379 was selected to screen out two candidate miRNAs:miR-103a-3p and miR-486-3p.(2)Compared with the control group,the expression level of miR-103a-3p in HK-2 cells transfected with overexpressed lentivirus was significantly decreased(P<0.01),and the expression level of miR-103a-3p in HK-2 cells transfected with circ＿0005379 siRNA was significantly increased(P<0.01).(3)The results of the double luciferase reporter experiment showed that the circ＿0005379 sequence was inserted into the double luciferase reporter gene plasmid,and the circ＿0005379 and miR-103a-3p mimics were co-transfected with circ＿0005379 and miR-103a-3p mimics.Compared with the NC group,the luciferase activity was significantly reduced.After the circ＿0005379 binding region was mutated and co-transfected with miR-103a-3p mimics,luciflucase activity did not change significantly.These results indicate that circ＿0005379 has binding effect with miR-103a-3p.(4)Fluorescence in situ hybridization results showed that circ＿0005379 was located in the cytoplasm of HK-2 cells,suggesting that circ＿0005379 might play a regulatory role through the mechanism of ce-RNA.(5)RNA immunoprecipitation assay results showed that,compared with the miR-103a-3p mimics control group,Ago2-bound circ＿0005379 level was significantly increased in HK-2 cells transfected with miR-103a-3p mimics(P<0.01).(6)The expression of miR-103a-3p was up-regulated in the HK-2 cell line cultured with high glucose,and the expression of miR-103a-3p was also found to be up-regulated in the peripheral blood of patients with diabetic nephropathy.2.Verification of binding relationship between miR-103a-3p and PTEN(1)The intersection of genes predicted by RNAhybrid,Targetscan and MiRanda software to combine with miR-103A-3p was selected to screen out four candidate genes:KLF4,PTEN,GPRC5A and TIMP3.(2)Compared with the control group,the expression of PTEN mRNA in cells transfected with miR-103a-3p mimic was significantly decreased(P<0.01),and the expression of PTEN mRNA in cells transfected with miR-103a-3p inhibitor was significantly increased(P<0.01).(3)After inserting PTEN-3’UTR sequence into double luciferase reporter plasmid and co-transfecting PTEN-3’UTR and miR-103a-3p mimic,luciferase activity was significantly decreased compared with NC group(P<0.05).After mutating the binding site of PTEN-3’UTR,luciferase activity in the group co-transfected with PTEN-3’UTR-MUT and miR-103a-3p mimic did not change significantly compared with the NC group.This indicates that there is a binding relationship between miR-103a-3p and PTEN.(4)The expression of PTEN was down-regulated in the HK-2 cell line cultured with high glucose,and the expression of PTEN in the peripheral blood of patients with diabetic nephropathy was down-regulated3.Circ＿00053 79 regulates the expression of PTEN(1)Compared with the control group,overexpression of circ＿0005379 group significantly increased the expression of PTEN mRNA in HK-2 cells(P<0.01).PTEN mRNA expression of HK-2 cells in circ＿0005379 group was significantly decreased(P<0.01).(2)Compared with the control group,the expression of PTEN protein in HK-2 cells in circ＿0005379 overexpression group was significantly increased(P<0.01),and that in circ＿0005379 knockdown group was significantly decreased(P<0.01).4＿HK-2 cells were transfected with miR-103a-3p inhibitor,and the cells were divided into four groups:NC group,HG group,HG+mir-103a-3p inhibitor contorl group,and HG+mir-103a-3p inhibitor group.QRT-PCR results showed that Compared with mir-103a-3p inhibitor contorl group,the expression level of mir-103a-3p inhibitor group was significantly decreased(P<0.01).The proliferation and apoptosis of HK-2 cells were detected by CCK8 method and apoptosis flow analysis,and the expression levels of fibrosis and autophagy related proteins were detected by western blot.(1)CCK8 results showed that HK-2 cell proliferation level in HG+miR103a-3p inhibitor group was significantly increased compared with HG+miR-103a-3p inhibitor contorl group(P<0.01).These results suggest that inhibiting miR-103a-3p expression could alleviate the glucotoxicity and significantly inhibit the apoptosis of HK-2 cells.(2)Flow cytometry results showed that compared with HG+miR-103a-3p inhibitor contorl group,total apoptosis levels of HK-2 cells in HG+mmiR-103a-3p inhibitor group were significantly decreased(P<0.01).These results suggest that inhibition of miR-103a-3p expression can alleviate glycotoxicity and significantly inhibit apoptosis of HK-2 cells.(3)Compared with HG+miR-103a-3p inhibitor contorl group,the expression levels of Col Ⅳ and a-SMA of fibrosis related proteins in HK-2 cells in HG+miR-103a-3p inhibitor group were significantly decreased(P<0.01).The ratio of autophagy related protein LC3Ⅱ/LC3Ⅰ was significantly increased and the expression of p62 was significantly decreased(P<0.01).It is suggesting that miR-103a-3p could affect the fibrosis and autophagy process of HK-2 cells.5.The overexpressed circ＿0005379 group was transfected with miR-103a-3p mimics,and the cells were divided into 3 groups:NC group,circ＿0005379+miR-103a-3p mimics control group,circ＿0005379+miR-103a-3p mimics control group.Compared with circ＿0005379+miR-103a-3p mimics control group,the expression level of miR-103a-3p in circ＿0005379+miR-103a-3p mimics control group was significantly increased(P<0.01).Compared with circ＿0005379+miR-103a-3p mimics control group,the expression level of PTEN protein in circ＿0005379+miR-103a-3p mimics control group was significantly decreased(P<0.01).6.The overexpressed circ＿0005379 group was transfected with miR-103a-3p mimics and si-PTEN.The cells were divided into four groups for rescue experiment:HG group,HG+circ＿0005379 group,HG+circ＿0005379+miR-103a-3p mimics group,HG+circ＿0005379+si-PTEN group,qRT-PCR results showed that compared with circ＿0005379 group,the expression level of PTEN protein in circ＿0005379+si-PTEN group was significantly decreased(P<0.01).(1)CCK8 results showed that HG+circ＿0005379+miR103a-3p mimics group,compared with HG+circ＿0005379 group,HK-2 cell proliferation was significantly decreased in HG+circ＿0005379+si-PTEN group(P<0.01).(2)Flow cytometry results showed that compared with the HG+circ＿0005379 group,the HG+circ＿0005379+miR-103a-3p mimics group,The levels of early apoptosis,late apoptosis and total apoptosis of HK2 cells in HG+circ＿0005379+si-PTEN group were significantly increased(P<0.01).(3)Western blot results showed that compared with the HG+circ＿0005379 group,the HG+ circ＿0005379+mir-103a-3p mimics group,HG+circ＿0005379+si-PTEN group,the Col Ⅳ and a-SMA expression levels of fibrosis related proteins in HK2 cells were significantly increased(P<0.01),and the ratio of autophagy related proteins LC3Ⅱ/LC3Ⅰ was significantly decreased.The expression ofp62 was significantly increased(P<0.01).ConclusionCirc＿0005379 binds to miR-103a-3p to regulate the expression of downstream target gene PTEN,promote the proliferation,inhibit apoptosis,alleviate fibrosis,and enhance autophagy of renal tubular epithelial cells,thus providing a new therapeutic method for the treatment of diabetic nephropathy. |
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