| Background:Idiopathic membranous nephropathy（IMN）is a common cause of the adult nephrotic syndrome.The incidence of IMN in our country is increasing in recent years.Without treatment in time,IMN will eventually irreversibly develop into chronic kidney disease or end-stage renal disease.At present,the diagnosis of IMN mainly relies on renal biopsy.Although the anti-PLA2R antibody is the best non-invasive marker at present,the positive rate is only 70-80%.Therefore,searching for new non-invasive diagnostic biomarkers of IMN is still the research focus.The pathogenesis of IMN is complex,and research on the mechanism of pathogenesis and development of IMN have become a hot and difficult topic in the field of kidney diseases.Exosomes are extracellular vesicles with a diameter of 40～160 nm,with good stability due to their double-layered membrane structure.Exosomes can contain various cellular components such as DNA,RNA,and proteins.In recent years,attention has been paid to the diagnostic potential of noncodingRNAs in exosomes.Among exosomal non-codingRNAs,circularRNAs（circRNAs）have a special ring structure,which makes them more stable than linearRNAs.The detection of urinary exosomal circRNAs is non-invasive,stable and specific,and has been shown to be biomarkers of various kidney diseases.With the continuous development ofRNA-sequencing（RNA-seq）technology,numerous studies have shown that circRNAs have unique expression profiles and important biological functions in various diseases.Through the analysis of the expression profile of circRNAs,the differentially expressed circRNAs in diseases can be screened,and their functions can be predicted and analyzed macroscopically,and then the molecular biomarkers of the disease and the signaling pathways related to the occurrence and development of the disease can be screened out,which could expand researchers’understanding of diseases at the molecular level.Previous studies have shown that circRNAs may be involved in the pathogenesis of IMN,but there are only a few related studies,and the specific mechanism of circRNAs in IMN is still unclear.Therefore,this research was established to further study circRNAs Possible role in IMN.Aims:The present study aims to investigate the profiling and functional prediction of urinary exosomal circRNAs of IMN patients and look for target urinary exosomal circRNAs as potential biomarkers for diagnosis of IMN patients and evaluate their diagnostic potential.Furthermore,we plan to conduct a preliminary study on the mechanism of target circRNAs in IMN.Methods:1.Expression profiling of circRNAs in IMN urinary exosomes Urine exosomes were collected from 5 IMN and 5 HCs by ultracentrifuge.A pairwise comparison between the two groups was performed by High-throughput sequencing.The size and shape of exosomes were detected by transmission electron microscopy,and the surface proteins of exosomes were detected by western blotting.Trizol reagent was used to extract totalRNA in urinary exosomes.After quality control,anRNA library was constructed,andRNA-sequencing（RNA-seq）was performed to analyze the differentially expressed circRNAs in urinary exosomes between the IMN group and HC group.Enrichment analysis including gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis were performed to explore the potential functions of the differentially expressed circRNAs in the IMN group.2.Preliminary study on the diagnostic potential of urinary exosome hsa＿circ＿0001250 Among the differentially expressed circRNAs,by searching circ Bank,we screened out the circRNAs with homology to mouse circRNAs,and screened out the first three circRNAs with original genes more related to kidney diseases and have a higher fold change in theRNA-sequencing results,they were hsa＿circ＿0004771,hsa＿circ＿0000896,and hsa＿circ＿0001250.23 IMN patients,19 patients with other idiopathic nephrotic syndrome（INS）and 23 HCs were enrolled in this study.TheRNAs in urinary exosomes were extracted,and the relative expression levels of hsa＿circ＿0004771,hsa＿circ＿0000896,and hsa＿circ＿0001250 among these three groups were compared by real-time quantitative reverse transcription PCR（q RT-PCR）experiments.hsa＿circ＿0001250 was finally identified as the target circRNA in this study.The circular structure of hsa＿circ＿0001250 was identified by Sanger sequencing of the fragment spanning the hsa＿circ＿0001250 splice site,RNase R digestion experiment followed by PCR product agarose gel electrophoresis and q RT-PCR.Sequence information and original genes of hsa＿circ＿0001250 were confirmed by Sanger sequencing.ROC curves were drawn to assess the diagnostic potential of hsa＿circ＿0001250.The correlation between hsa＿circ＿0001250 and clinical indicators of IMN patients was analyzed by Pearson correlation analysis.By constructing the circRNA-miRNA-mRNA network of hsa＿circ＿0001250,the possible signal pathways and biological functions of hsa＿circ＿0001250 were predicted.3.Preliminary exploration of the role of hsa＿circ＿0001250 in IMN Angiotensin II（Ang II）was used to construct a human glomerular podocyte injury model.Cell apoptosis was detected by flow cytometry,measurement of caspase 3enzyme activity,and caspase 9 enzyme activity.The expression of NLRP3 and cleaved caspase 1 protein was detected by WB,and the expression of IL-1βin cell supernatant was detected by ELISA to detect cell inflammation.TheRNA was isolated and purified by nucleocytoplasmic separation technology,and the subcellular localization of hsa＿circ＿0001250 was detected by a q RT-PCR experiment.hsa＿circ＿0001250 was knocked down by small interferingRNA（siRNA）.The expression levels of hsa＿circ＿0001250 and hsa＿mi R-876-5p in the injury model were detected by q RT-PCR experiments.The binding of the two was detected by a luciferase reporter gene assay.The expression of hsa＿mi R-876-5p was down-regulated or up-regulated by interference（inhibitor）and overexpression（mimics）vectors.Flow cytometry,measurement of caspase 3 enzyme activity,measurement of caspase 9 enzyme activity,WB detection of NLRP3,cleaved caspase 1 protein expression,ELISA detection of IL-1βexpression in cell supernatant,were detected to confirm that hsa＿circ＿0001250 promotes Ang II-induced podocyte injury by targeting hsa＿mi R-876-5p.Results:1.Expression profiling of urinary exosomal circRNAs in IMN We identified 766 up-regulated and 283 down-regulated circRNAs in IMN patients,among which 461 circRNAs were identified for the first time.The length of up-regulated circRNAs is mostly<500nt,and the length of down-regulated circRNAs is mostly 500-2000nt.The differentially expressed circRNAs were distributed in autosomes,sex chromosomes,and mitochondrial chromosomes,and the down-regulated circRNAs were mostly derived from mitochondrial DNA.The up-regulated circRNAs were mostly derived from exons,and the down-regulated circRNAs were mostly derived from introns.The results of GO analysis showed that the up-regulated circRNAs may be involved in the regulation of NF-κB signaling pathway and protein ubiquitination,while the downregulated circRNAs may be involved in the apoptosis process.KEGG analysis indicated that up-regulated circRNAs were involved in endocytosis and protein transport pathways in the endoplasmic reticulum,and down-regulated circRNAs were involved in Notch signaling pathway,AMPK signaling pathway,and RIG-I-like receptor signaling pathway.2.Preliminary study on the diagnostic potential of urinary exosome hsa＿circ＿0001250 Among the differentially expressed circRNAs screened byRNA high-throughput sequencing,we confirmed that the expression of urinary exosomal hsa＿circ＿0001250was significantly increased in the IMN group,but there was no difference in the expression of hsa＿circ＿0004771 or hsa＿circ＿0000896 among the three groups.We confirmed the ring structure of hsa＿circ＿0001250,and confirmed that the sequence of hsa＿circ＿0001250 is consistent with the information in circ Base database,formed by back splicing of exon 2 and exon 3 of GRAMD4.ROC curve analysis confirmed the efficacy of hsa＿circ＿0001250 in diagnosing IMN,the area under the curve was 0.8034（95%CI:0.6667-0.9401）.hsa＿circ＿0001250 also could assist in the differential diagnosis of IMN from other INS,and the area under the curve was 0.7872.Pearson correlation analysis showed that the expression of hsa＿circ＿0001250 was positively correlated with proteinuria（r=0.6402,P<0.01）.A circRNA-miRNA-mRNA network was constructed.3.Preliminary exploration of the mechanism of action of hsa＿circ＿0001250 The podocytes were incubated for 24 h with the complete medium of Ang II solution with a concentration of 10-7 mol/L.Results of Flow cytometry showed that the apoptosis rate increased,and the enzyme activity measurement showed that the activity of caspase 3 and caspase 9 increased,indicating that Ang II can induce podocyte apoptosis.Results of WB showed that the expression of NLRP3 and cleaved caspase 1increased,and ELISA showed that the expression of IL-1βin the cell supernatant increased,confirming that Ang II can activate the NLRP3 inflammasome and cause podocyte inflammatory injury.Nucleocytoplasmic separation experiments confirmed that hsa＿circ＿0001250 was mostly expressed in the cytoplasm.In podocytes,after hsa＿circ＿0001250 was down-regulated by siRNA,the expression of hsa-mi R-876-5p was up-regulated.It was predicted and confirmed by luciferase reporter gene experiment that hsa＿circ＿0001250 could bind to hsa-mi R-876-5p.In the Ang II-induced podocyte injury model,hsa＿circ＿0001250 was up-regulated and promoted podocyte injury,and hsa-mi R-876-5p was down-regulated and inhibited podocyte injury.After down-regulating hsa＿circ＿0001250 expression with siRNA,podocyte damage was reduced,and this process could be rescued by down-regulating hsa＿mi R-876-5p expression.Therefore,this study suggests that hsa＿circ＿0001250 promotes Ang II-induced podocyte apoptosis and NLRP3 inflammasome activation-induced inflammatory injury through sponging and down-regulating the expression of hsa＿mi R-876-5p.Conclusion:1.We revealed the expression and functional profile of differentially expressed urinary exosomal circRNAs in IMN patients.2.Urinary exosome hsa＿circ＿0001250 showed potential to be a non-invasive biomarker of IMN,with a significant positive correlation with proteinuria.3.In this study,a podocyte injury model was constructed by Ang II,and it was found that the highly expressed hsa＿circ＿0001250 can sponge and down-regulate the expression of hsa＿mi R-876-5p,thereby promoting Ang II-induced podocyte apoptosis and inflammatory injury caused by NLRP3 inflammasome,which preliminarily clarified the mechanism of hsa＿circ＿0001250 involved in the occurrence and development of IMN. |
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