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Nephrology
Basic research

Differential expression study of circular RNAs in exosomes from serum and urine in patients with idiopathic membranous nephropathy

Hualin Ma, Ying Xu, Rongrong Zhang, Baochun Guo, Shuyan Zhang, Xinzhou Zhang

Arch Med Sci 2019; 15 (3): 738–753
Online publish date: 2019/04/30
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Introduction

Idiopathic membranous nephropathy (IMN) is the most common cause of adult nephrotic syndrome. Approximately 25% to 40% of adult primary nephrotic syndrome cases have IMN. Idiopathic membranous nephropathy is also the most common pathologic type of glomerular disease, and IMN has a longer disease course. The prognosis of IMN varies [1]. The pathologic features of IMN are a high number of immune complexes deposited in the glomerular basement membrane on the epithelium side.

The exosome has a double layer plasma membrane structure. Its diameter is approximately 30–100 nm, and it carries a rich protein, mRNA and microRNA. Exosomes are released to the extracellular microenvironment by the cells [2, 3]. They can be released from fibroblasts, dendritic cells, tumor cells and other cells; they are widespread in the urine [4], peripheral blood, saliva, cerebrospinal fluid, amniotic fluid, ascites and other body fluids [3, 5]. Therefore, we can detect exosomes and their contents from tissue, cells and body fluids to diagnose and clinically treat the disease, especially kidney disease. Miranda et al. [6] observed exosomes of renal tubular epithelial cells, podocytes, collecting duct cells and leap cells by transmission electron microscopy, which showed that almost all kidney inherent cells could secrete exosomes. In addition, the authors found that the components of exosomes were different in normal physiological conditions and disease conditions even for the same tissue or body fluid [7]. Previous studies have shown that the contents of exosomes have a characteristic change in acute kidney injury [8], IgA nephropathy [9], diabetic nephropathy [10], renal tubular acidosis [6], polycystic kidney [11] and other kidney diseases. The findings suggested that exosomes can be used as specific markers for early disease diagnosis.

Recent studies have shown that circRNAs can be used as biomarkers for the diagnosis and efficacy of a variety of clinical diseases, such as atherosclerosis [12], neurological diseases [1315], diabetes [16], tumors [1719] and more. In addition, because of the high stability of the circRNAs and the difficulty of degrading them by exonuclease, we can easily obtain circRNA from body fluid [12]. Based on the above characteristics, circRNAs show great potential to regulate human disease genes [20], making them a current research focus. In 2005, Huang found many exosomes in human serum and discovered that there is a difference in the exo-circRNA between colorectal cancer and normal human serum [21]. The authors speculated that circRNAs could be used as a new biomarker for cancer diagnosis. This discovery renewed people’s awareness of circRNAs and exosomes because the authors had linked two emerging areas and further demonstrated the importance of circRNA and exosomes in organisms [21].

In this study, we evaluated circRNAs of exosomes. We compared the expression of circRNAs in the exosomes of serum and urine in patients with idiopathic membranous nephropathy and normal healthy controls by gene sequencing. Then, we screened out the differential expression of circRNAs and performed further analysis. The rich data from the analysis provide insight into the pathogenesis of IMN and a solution for future diagnosis and treatment.

Material and methods

Patient assessments and classifications

The study protocols and consent forms were approved by the Second Clinical Medical College (Shenzhen People’s Hospital) of Jinan University and adhere to the Helsinki Declaration guidelines on ethical principles for medical research involving human subjects. Written informed consent was obtained from all participants. Ten IMN patients who had never been treated with glucocorticoids or other immunosuppressive drugs were recruited for this study. In addition, we chose 10 healthy subjects as controls (Table I).

Table I

Clinical characteristics of IMN patients and normal controls

GroupIMN groupNC group
Age [years]38.61 ±11.2135.14 ±12.13
Sex (M/F)7/37/3
Serum creatinine [µmol/l]74.9 ±23.663.8 ±20.4
Proteinuria [g/24 h]2.50 ±1.280.08 ±0.03
Serum albumin [g/l]34.04 ±8.7942.57 ±3.16
PLA2R (%)60% (6/10)0% (0/10)

[i] NC group – normal control group.

Inclusion and exclusion criteria

The inclusion criteria were as follows: IMN patients were hospitalized at Shenzhen People’s Hospital nephrology department from November 2015 to October 2016. Renal biopsy confirmed that their pathological type was idiopathic membranous nephropathy and their kidney function was normal before and after admission.

The exclusion criteria were as follows: 1) patients with abnormal renal function based on increased urea nitrogen or creatinine; 2) secondary nephrotic syndrome patients, such as those with hypertensive nephropathy, diabetic nephropathy, lupus nephritis, and hepatitis-related nephritis; and 3) renal pathology results confirming membranous nephropathy, but the patient has co-occurrence of another disease that can cause renal damage, such as hypertension, diabetes, systemic lupus erythematosus, hepatitis B and others.

Collection of serum and urine specimens:

  1. All patients met the inclusion criteria and they were prohibited from eating or drinking the night before specimens were collected.

  2. Venous blood was collected the next morning from elbow vein blood and then kept at 37°C to promote coagulation.

  3. Samples were centrifuged for approximately 10 min at 3000 rpm.

  4. Approximately 2–3 ml of the upper layer of liquid was absorbed into the EP tube, which was marked with identification information (date, number, etc.) and then stored at –80°C.

  5. At the same time, the patient’s first morning urine (approximately 100 ml) was collected into a centrifuge tube, which was marked with identifying information (date, number, etc.) and then stored at –80°C.

Exosome isolation

Exosomes were isolated by the polymer formulation method [22] from blood serum using an ExoQuick reagent precipitation kit (System Biosciences, SBI, Mountain View, CA) according to the manufacturer’s protocol. This exosome isolation method has been well validated with other techniques, including electron microscopy [22, 23]. All exosomes were stored at –80°C immediately after isolation until further analysis. The total protein concentration of the isolated exosomes was determined using the standard Bradford protein assay (Bio-Rad, Richmond, VA, USA).

Isolation of RNA from exosomes

Exosome supernatants were added to 40 pM synthetic cel-miR-39 (UCACCGGGUGUAAAUCAGCUUG) to control and normalize the efficiency of RNA extraction; then, they were transferred to RNase-free tubes for RNA isolation using an miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The RNA sample was washed twice in 500 µl of RPE buffer and eluted in RNase-free water. The isolated RNA was measured using a NanoDrop 1000 ultraviolet spectrophotometer (Thermo Fisher Scientific) and analyzed by reverse transcription polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR).

Serum and urine exosome circRNA sequencing

The total RNA was extracted and then was digested with DNase I to remove rRNA; then, RNase R was used to remove the linear RNA, enriching the circRNAs. The circRNAs were fragmented, and the first strand cDNA was synthesized by reverse transcription using random primers. Then, the second strand cDNA was synthesized using dNTP containing dUTP. The secondary chain product was subjected to terminal repair, and was pulsed “A” and a linker. The reaction mixture was digested with USER enzyme to remove the second strand cDNA containing dUTP, and a primer was added to amplify via PCR and obtain a chain-specific cDNA library. The fragments were screened by magnetic beads. Quality control was performed and fragments were further sequenced on a machine. The experimental procedure is summarized in Figure 1.

Figure 1

The main experimental process of circular RNA gene sequencing. QC1: detection of the total RNA concentration, purity, completeness; QC2: confirmed that more than 99% of the rRNA had been removed; QC3: confirmed that RNA was fragmented into approximately 200-bp fragments; QC4: detected the fragment concentration and size and library concentration. The distribution of the significantly differentially expressed miRNA of cells in the cellular component with high throughput sequencing

/f/fulltexts/AOMS/36465/AMS-15-36465-g001_min.jpg

Bioinformatics analysis

The expression values calculated for the differential proteins and peptides were used for the distance and average to determine the linkage for gene ontology (GO) analysis. In pathway analysis, interactions between genes in the range of genomes were analyzed by downloading the pathway data in KEGG. Finally, the results of the above data were merged into a comprehensive gene inter-relationship network. The established gene network could directly reflect the inter-relationships between genes at a whole-cell level as well as the stability of the gene regulatory network.

Statistical analysis

The back-spliced junction reads and linear mapped reads were combined and scaled to reads per million mapped reads (RPM) to quantify circRNA expression levels. Differences in circRNA expression levels were analyzed using Student’s t-test. P < 0.05 was considered statistically significant.

Results

Total RNA quality and concentration determination results

RNA was extracted and purified using an RNA isolation kit. The total RNA of the IMN and NC groups was detected with a Qubit3.0 fluorescence meter. The results are shown in Tables II and III. In the tables, the total amount of exosome RNA measured in each group was more than 200 ng, and the obtained circRNAs had high purity and good integrity, and could be used for later experiments.

Table II

Concentration of exosome total RNA

Sample nameSerum volume [ml]Exosome RNA concentration [ng/µl]Exosome RNA total amount [ng]
IMN group28.79.69242.30
NC group2333.40400.80

[i] IMN group – IMN group, NC group – normal control group.

Table III

Concentration of exosome total RNA

Sample nameUrine volume [ml]Exosome RNA concentration [ng/µl]Exosome RNA total amount [ng]
IMN group98016.50445.50
NC group97016.70367.40

[i] IMN group – IMN group, NC group – normal control group.

Types of circRNAs

Compared with the healthy control group, the types of circRNAs in the serum of the patients with idiopathic membranous nephropathy decreased and mainly appeared as intron region sources. However, the circRNAs in the urinary exosomes increased, and mainly appeared to have an exon region source (Table IV).

Table IV

Species of exosome circRNA

VariableIMN serumIMN urineNC serumNC urine
Total number of circRNAs8528622712
Number of circRNAs from the circBase database019861
Number of circRNAs from the exon region5218122
Number of circRNAs from the intron region60581896
Number of circRNAs from the intergenic region2010261

[i] IMN group – IMN group, NC group – normal control group.

Difference analysis of circRNAs

According to the expression level of circRNAs, when the difference multiple (ratio) was more than 2 or less than 0.5 and FDR ≤ 0.001, the circRNAs were considered differentiated. In this study, the log2 ratio was used instead of multiple differences. The filter criteria of significantly differentially expressed genes were FDR ≤ 0.001 and |log2 ratio| ≥ 1.

Differential expression of circRNAs in serum and urine exosomes of IMN patients

According to the experimental results, there were 59 species of circRNA with significantly different expression compared to serum and urine exosomes in IMN patients; 32 species were up-regulated (Table V) and 27 species were down-regulated (Table VI). Most of these circRNAs had an intron source. The corresponding genes were mainly SNORA25, SNORA31, SNORA51, SNORA75 and other nucleolus small RNAs. The log2 ratio of chrY: 13688616|13833086 was 27.592 in the up-regulation circRNA, which was the most significant. The log2 ratio of chrY:13842647|13855594 was –26.379 in the down-regulation circRNA, which was the most significant. However, the two most significantly different circRNAs in the circBase gene pool had no corresponding gene, suggesting that they may be newly discovered genes.

Table V

Up-regulated circRNAs between the IMN serum and IMN urine

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chrY:13688616|1383308627.592UpIntergenic regionchrYn/a
chrY:13650802|1372592126.677UpIntergenic regionchrYn/a
chrY:13844080|1385174125.449UpIntergenic regionchrYn/a
chr3:114874721|11487473924.936UpIntronchr3SNORA25
chr4:49133318|4915181224.363UpIntronchr4SNORA75
chr4:49133318|4915181723.766UpIntronchr4SNORA75
chr1:246981249|24698130823.51UpIntronchr1SNORA25
chr6:39390231|3939025123.444UpIntronchr6KIF6
chrY:13688616|1381031823.251UpIntergenic regionchrYn/a
chrY:13867301|1386948623.116UpIntergenic regionchrYn/a
chr3:42154842|4215488923.059UpIntronchr3TRAK1
chr8:70602312|7060240922.531UpIntronchr8SLCO5A1
chr4:49118019|4912872222.398UpIntronchr4SNORA51
chr10:39103465|3910572622.302UpIntronchr10SNORA31
chr8:70602353|7060243122.029UpIntronchr8SLCO5A1
chr8:70602360|7060242721.967UpIntronchr8SLCO5A1
chr10:39085864|3908829521.903UpIntronchr10SNORA31
chr1:91853081|9185313921.766UpIntronchr1SNORA31
chrY:13684026|1384407921.614UpIntergenic regionchrYn/a
chr3:114874721|11487474321.614UpIntronchr3SNORA25
chr8:70602355|7060242721.531UpIntronchr8SLCO5A1
chr4:49637530|4964186721.351UpIntronchr4SNORA51
chr4:49120156|4912108421.351UpIntronchr4SNORA51
chr8:70602312|7060238221.351UpIntronchr8SLCO5A1
chr17:22246001|2225330121.351UpIntronchr17snoU13
chr21:10778969|1080832621.251UpIntronchr21SNORA70
chr1:108113527|10811359521.251UpIntronchr1SNORA31
chrY:13659053|1384407921.144UpIntronchrYn/a
chr15:101250552|10125065321.144UpIntronchr15snoU13
chr21:44593818|4459390321.144UpIntergenic regionchr21n/a
chr7:100550808|1005510624.267UpExonchr7MUC3A
chrY:13805036|138411343.876UpIntergenic regionchrYn/a

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Table VI

Down-regulated circRNAs between the IMN serum and IMN urine

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chrY:13842647|13855594–26.379DownIntergenic regionchrYn/a
chrY:13650802|13659298–26.116DownIntergenic regionchrYn/a
chr17:22248380|22253301–25.516DownIntronchr17snoU13
chr8:43092760|43093139–25.146DownIntronchr8SNORD112
chr8:43092873|43096758–24.588DownIntronchr8SNORD112
chr4:90986390|90986415–24.441DownIntronchr4SNORA51
chr4:49103783|49111822–23.791DownIntronchr4SNORA51
chr10:39139428|39147131–23.244DownIntronchr10SNORA31
chr4:49641376|49652154–23.221DownIntronchr4SNORA51
chr18:54265993|54266355–23.079DownExonchr18TXNL1
chr6:158779108|158779264–22.894DownIntronchr6TULP4
chr2:19441309|19442090–22.806DownIntronchr2SNORA51
chr3:96221435|96221837–22.776DownIntronchr3SNORA25
chrY:13801063|13849765–22.266DownIntergenic regionchrYn/a
chr8:43095798|43096720–21.976DownIntronchr8SNORD112
chr6:61899754|61913064–21.806DownIntronchr6SNORD45
chr8:43093689|43097076–21.681DownIntronchr8SNORD112
chr2:233244474|233272478–21.614DownIntronchr2snoU13
chr2:221311242|221311332–21.543DownIntronchr2SNORA75
chr20:30954187|30956926–21.543DownExonchr20ASXL1
chr4:35172567|35172590–21.469DownIntronchr4SNORA75
chr6:2024936|2340390–21.391DownIntronchr6snoU13
chr10:18831781|18831900–21.309DownIntronchr10SNORA31
chr10:42400571|42533897–21.221DownIntronchr10SNORA31
chr15:30465080|30465505–21.128DownIntronchr15SNORA48
chr19:34882415|34883413–3.063DownIntronchr19GPI
chrY:13691698|13851741–1.454DownIntergenic regionchrYn/a

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Differential expression of circRNAs in serum exosomes of IMN and NC patients

According to the experimental results, there were 89 species of circRNAs with significantly different expression compared to IMN patients’ serum exosomes and NC patients’ serum exosomes; 49 species were up-regulated (Table VII) and 40 species were down-regulated (Table VIII). Most of these circRNAs had an intron source. The corresponding genes were mainly SNORA25, SNORA51, SNORA31, SNORA75, SNORD112 and other nucleolus small RNAs. The log2 ratio of chrY:13688616|13833086 was 27.592 in the up-regulation circRNAs, which was the most significant. However, the circRNAs in the circBase gene pool had no corresponding gene, which suggested that it may be a newly discovered gene. The log2 ratio of chr2:233244474|233272478 was –27.111 in the down-regulation circRNAs, which was the most significant, and the corresponding gene is the snoU13 gene. This gene is mainly expressed nucleolus small RNA and plays a role in RNA treatment and modification.

Table VII

Up-regulated circRNAs between the IMN and NC groups in serum

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chrY:13688616|1383308627.592UpIntergenic regionchrYn/a
chrY:13650802|1372592126.677UpIntergenic regionchrYn/a
chr3:114874721|11487473924.936UpIntronchr3SNORA25
chr4:49133318|4915181224.363UpIntronchr4SNORA51
chr4:49133318|4915181723.766UpIntronchr4SNORA51
chr6:39390231|3939025123.444UpIntronchr6KIF6
chrY:13688616|1381031823.251UpIntergenic regionchrYn/a
chr3:42154842|4215488923.059UpIntronchr4TRAK1
chr4:49118019|4912872222.398UpIntronchr4SNORA51
chr10:39103465|3910572622.302UpIntronchr10SNORA31
chr10:39085864|3908829521.903UpIntronchr10SNORA31
chr1:91853081|9185313921.766UpIntronchr1HFM1
chrY:13684026|1384407921.614UpIntergenic regionchrYn/a
chr3:114874721|11487474321.614UpIntronchr3SNORA25
chr8:70602355|7060242721.531UpIntronchr8SLCO5A1
chr4:49637530|4964186721.351UpIntronchr4SNORA51
chr4:49120156|4912108421.351UpIntronchr4SNORA51
chr17:22246001|2225330121.351UpIntronchr17snoU13
chr21:10778969|1080832621.251UpIntronchr21SNORA70
chr1:108113527|10811359521.251UpIntronchr1SNORA51
chrY:13659053|1384407921.144UpIntergenic regionchrYn/a
chr15:101250552|10125065321.144UpIntronchr15snoU13
chr21:44593818|4459390321.144UpIntergenic regionchr21n/a
chr2:5845511|584595420.766UpIntronchr2snoU13
chr4:70296654|7029671020.614UpIntronchr4SNORA51
chr7:71387989|7138802720.614UpIntronchr7CALN1
chr16:47538682|4753875420.614UpIntronchr16PHKB
chr19:34882415|3488341320.444UpIntronchr19GPI
chr2:92305623|9230935820.444UpIntronchr2SNORA75
chr20:59906635|5990677620.251UpIntronchr20CDH4
chr1:91852914|9185299620.029UpIntronchr1HFM1
chr10:38778641|3881658120.029UpIntronchr10SNORA31
chrX:108297654|10829770920.029UpExonchrXCTD-2328D6.1
chr12:38237430|3850295120.029UpIntronchr12SNORD112
chr20:59906715|5990677619.766UpIntronchr20CDH4
chr14:70396886|7039695419.766UpIntronchr14SMOC1
chr8:70602368|7060243119.766UpIntronchr8SLCO5A1
chr10:51358680|5163606719.766UpIntronchr10SNORA31
chr8:70602312|7060242019.766UpIntronchr8SLCO5A1
chr8:70602360|706024274.413UpIntronchr8SLCO5A1
chr1:246981249|2469813083.955UpIntronchr1SNORA25
chr7:100550808|1005510623.349UpExonchr7MUC3A
chrY:13867301|138694862.807UpIntergenic regionchrYn/a
chrY:13805036|138411342.806UpIntergenic regionchrYn/a
chr8:70602353|706024312.567UpIntronchr8SLCO5A1
chrY:13691698|138517412.522UpIntergenic regionchrYn/a
chr8:70602312|706024092.276UpIntronchr8SLCO5A1
chrY:13844080|138517412.012UpIntergenic regionchrYn/a
chrY:13688616|138516911.936UpIntergenic regionchrYn/a

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Table VIII

Down-regulated circRNA between the IMN and NC groups in serum

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chr2:233244474|233272478–27.111DownIntronchr2snoU13
chr17:39537965|39552828–26.966DownIntronchr17SCARNA20
chr22:42910112|42970824–26.895DownIntronchr22Y_RNA
chr6:31122297|31122344–26.054DownExonchr6CCHCR1
chr12:52863454|52909616–25.798DownIntronchr12SNORD112
chr19:36066505|36066634–25.772DownIntronchr19SNORA70
chr4:1005136|1242947–25.454DownIntergenic regionchr4n/a
chr4:159973545|159973572–25.028DownIntronchr4SNORA51
chrY:13842647|13855594–24.668DownIntergenic regionchrYn/a
chr21:37558665|37558690–22.588DownIntronchr21DOPEY2
chr15:31645251|31645272–22.461DownIntronchr15KLF13
chr10:39139428|39141998–22.387DownIntronchr10SNORA31
chr17:39938846|39938869–22.336DownIntronchr17JUP
chr17:22253135|22260437–22.198DownIntronchr17snoU13
chr17:79502678|79502749–21.981DownIntronchr17FSCN2
chr17:48266264|48272839–21.912DownExonchr17COL1A1
chr10:38804894|38818467–21.894DownIntronchr10SNORA31
chr1:74953936|74953971–21.858DownIntronchr1TMEM56
chr5:116075463|116075487–21.764DownIntronchr5SNORA70
chr18:18518121|18519655–21.764DownIntronchr18SNORD112
chr7:148028455|148028529–21.744DownIntronchr7CNTNAP2
chr2:189121958|189121979–21.724DownIntronchr2SNORA48
chrX:3349826|3349848–21.642DownIntronchrXsnoU13
chr11:75979847|75979884–21.599DownIntronchr11SNORA1
chr15:42134880|42134903–21.509DownExonchr15PLA2G4B
chr18:32291302|32291329–21.387DownIntronchr18DTNA
chr13:36337738|36337787–21.362DownIntronchr13SNORA25
chr17:31559413|31559527–21.336DownIntronchr17ASIC2
chr9:19592476|19592555–21.282DownIntronchr9SLC24A2
chr7:76626497|76626556–21.282DownIntronchr7DTX2P1
chr8:124924619|124924638–21.198DownIntronchr8FER1L6
chr1:7769121|7769144–21.078DownIntronchr1CAMTA1
chr14:37211610|37211628–21.046DownIntronchr14SLC25A21
chr1:32294226|32294254–20.947DownIntronchr1SNORA70
chr17:31559408|31559527–20.912DownIntronchr17ASIC2
chr19:56438931|56438947–20.84DownIntronchr19NLRP13
chr1:155048684|155048737–20.764DownIntronchr1EFNA3
chr20:46681136|46681159–20.764DownIntronchr20snoU13
chr1:233454768|233454781–20.764DownIntronchr1SNORA25
chr6:104238460|104238484–3.592DownIntronchr6SNORA33

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Differential expression of circRNAs in urine exosomes of IMN and NC patients

According to the experimental results, there were 60 species of circRNAs with significantly different expression compared to IMN patients’ urine exosomes and NC patients’ urine exosomes; 54 species were up-regulated (Table IX) and 6 species were down-regulated (Table X). Approximately 55% were intron sources, 30% were exon sources and 15% were intergenic regions. The corresponding genes were mainly SNORA51, SNORA31, SNORA70, SNORA75, SNORD112 and other nucleolus small RNAs. The log2 ratio of chrY:13842647|13855594 was 26.379 in the up-regulated circRNA, which was the most significant. The log2 ratio of chrY:13688616|13833086 was –25.049 in the down-regulated circRNAs, which was the most significant. However, the two most significantly different circRNAs in the circBase gene pool had no corresponding gene, suggesting that they may be newly discovered genes.

Table IX

Up-regulated circRNAs between the IMN and NC groups in urine

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chrY:13842647|1385559426.379UpIntergenic regionchrYn/a
chrY:13691698|1385174126.006UpIntergenic regionchrYn/a
chr17:22248380|2225330125.516UpIntronchr17snoU13
chr8:43092760|4309313925.146UpIntronchr8SNORD112
chr8:43092873|4309675824.588UpIntronchr8SNORD112
chr4:49103783|4911182223.791UpIntronchr4SNORA51
chr19:34882415|3488341323.507UpIntronchr19GPI
chr10:39139428|3914713123.244UpIntronchr10SNORA31
chr4:49641376|4965215423.221UpIntronchr4SNORA51
chr18:54265993|5426635523.079UpExonchr18TXNL1
chr6:158779108|15877926422.894UpIntronchr6TULP4
chr2:19441309|1944209022.806UpIntronchr2SNORA51
chr3:96221435|9622183722.776UpIntronchr3SNORA25
chrY:13801063|1384976522.266UpIntergenic regionchrYn/a
chr8:43095798|4309672021.976UpIntronchr8SNORD112
chr6:61899754|6191306421.806UpIntronchr6SNORD45
chr8:43093689|4309707621.681UpIntronchr8SNORD112
chr2:233244474|23327247821.614UpIntronchr2snoU13
chr2:221311242|22131133221.543UpIntronchr2SNORA75
chr20:30954187|3095692621.543UpExonchr20ASXL1
chr4:35172567|3517259021.469UpIntronchr4SNORA75
chr6:2024936|234039021.391UpIntronchr6snoU13
chr10:18831781|1883190021.309UpIntronchr10SNORA31
chr10:42400571|4253389721.221UpIntronchr10SNORA31
chr15:30465080|3046550521.128UpIntronchr15SNORA48
chr21:10788458|1085376221.029UpIntronchr21SNORA70
chr18:18518121|1851965520.806UpIntronchr18SNORD112
chrY:13805036|1384113420.806UpIntergenic regionchrYn/a
chr3:196118684|19612989020.681UpExonchr3UBXN7
chr5:137320946|13732400420.543UpExonchr5FAM13B
chr9:137976113|13797620720.543UpIntronchr9OLFM1
chr4:49101961|4915530620.543UpIntronchr4SNORA75
chr11:33307959|3330905720.391UpExonchr11HIPK3
chr17:20107646|2010922520.221UpExonchr17SPECC1
chr19:7034465|703616120.221UpIntronchr19Y_RNA
chr8:141874411|14190086820.029UpExonchr8PTK2
chr8:99718695|9971953920.029UpExonchr8STK3
chr13:64330137|6439806020.029UpIntronchr13SNORA25
chr8:43093228|4309707620.029UpIntronchr8SNORD112
chrY:13140123|1345695320.029UpIntergenic regionchrYn/a
chr21:16386665|1641589519.806UpExonchr21NRIP1
chr10:32197100|3219949119.806UpExonchr10ARHGAP12
chr14:76633006|7666231519.806UpExonchr14GPATCH2L
chrY:13137990|1345001919.806UpIntergenic regionchrYn/a
chr21:10789780|1083671719.543UpIntronchr21SNORA70
chr2:61749746|6176103819.543UpExonchr2XPO1
chr5:72370569|7237332019.543UpExonchr5FCHO2
chr1:180953813|18096256119.543UpExonchr1STX6
chr2:228252617|22825264319.543UpIntronchr2SNORA75
chr2:202010101|20201455819.543UpExonchr2CFLAR
chr20:52773708|5278820919.543UpExonchr20CYP24A1
chr9:113734353|11373583819.543UpExonchr9LPAR1
chr6:4891947|48926134.346UpExonchr6CDYL
chrY:13650802|136592981.893UpIntergenic regionchrYn/a

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Table X

Down-regulated circRNAs between the IMN and NC groups in urine

circRNALog2 ratioUp/downCircRNA typeChromosome localizationGene localization
chrY:13688616|13833086–25.049DownIntergenic regionchrYn/a
chr17:25267933|25267961–23.323DownIntronchr17snoU13
chr10:39084961|39105726–23.142DownIntronchr10SNORA31
chr10:38787997|39138199–22.485DownIntronchr10SNORA31
chr14:105944010|105944069–20.583DownIntronchr14CRIP2
chrY:13688616|13851691–1.126DownIntergenic regionchrYn/a

[i] NB: Gene ID n/a indicates that there was no matched circRNA in the circBase gene bank.

Bioinformatics analysis

Target genes were analyzed for their potential functions using GO and KEGG pathways. GO analysis demonstrated that the target genes were associated with cellular processes, multicellular organisms, pigmentation, the development process and the response to stimuli at both serum and urine exosomes (Figures 2 and 3). Furthermore, significantly associated pathways comprising the target genes were obtained for the assessed circRNAs. Interestingly, we selected 29 metabolic pathways in the serum sample; of all 29 pathways, 21 had PLA abnormalities, and the corresponding gene was PLA2G4B. The top 20 signaling pathways are shown in Figure 4, while the platelet activation signaling pathway was the most widely distributed (Figure 5). In addition, we selected 35 metabolic pathways in the urine samples. The top 20 are shown in Figure 6, while the P13K-Akt signaling pathway was the most widely distributed (Figure 7).

Figure 2

GO annotation of differentially expressed circRNAs in the serum exosomes of IMN patients compared to the control group. GO annotation consisted of the biological process, cellular components, and molecular function

/f/fulltexts/AOMS/36465/AMS-15-36465-g002_min.jpg
Figure 3

GO annotation of differentially expressed circRNAs in urine exosomes of IMN patients compared to the control group. GO annotation consisted of the biological process, cellular component, and molecular function

/f/fulltexts/AOMS/36465/AMS-15-36465-g003_min.jpg
Figure 4

KEGG pathway analysis of predicted targets for differentially expressed circRNAs in serum exosomes of IMN patients compared to the control group. The bluer the circle, the more significant the pathway enrichment. The bigger the circle, the higher the number of pathway genes

/f/fulltexts/AOMS/36465/AMS-15-36465-g004_min.jpg
Figure 5

Pathway analysis of differential genes: platelet activation. Red marks indicate the genes with differential profiles

/f/fulltexts/AOMS/36465/AMS-15-36465-g005_min.jpg
Figure 6

KEGG pathway analysis of predicted targets for differentially expressed circRNAs in urine exosomes of IMN patients compared to the control group. The bluer the circle, the more significant the pathway enrichment. The bigger the circle, the higher the number of pathway genes

/f/fulltexts/AOMS/36465/AMS-15-36465-g006_min.jpg
Figure 7

Pathway analysis of differential genes: the PI3K-Akt signaling pathway. Red marks indicate the genes with differential profiles

/f/fulltexts/AOMS/36465/AMS-15-36465-g007_min.jpg

Discussion

Beck et al. [24] detected anti-PLA2R antibodies for the first time in IMN patient plasma samples. Substantial clinical data showed its specificity of up to 100% and sensitivity of approximately 70% to 80%, which indicated that they can be used as IMN-specific diagnostic markers.

The latest study [24, 25] showed that the mannose-binding lectin pathway was the major complement activation in the pathogenesis of IMN. In this study, the mucin 3A (MUC3A) gene, corresponding to the circRNAs of chromosome 7 encoding chr7: 100550808|100551062 in the serum exosomes of IMN patients, was significantly up-regulated. It was also found that MUC3A was encoded by an exon-derived gene. Existing studies have shown that [26] MUC3A is a mucin cluster located on the 7p22 chromosome. Additionally, MUC3A belongs to a transmembrane glycoprotein. Authors [27] found that 71% of the amino acid repeated sequences encoded by MUC3 were serine/threonine and 6% proline. Studies have demonstrated that the activation of serine proteases is achieved by a change in specific amino acid residues in the center of serine-dominated activity [28]. Because most of the amino acids encoded by the MUC3A gene in this study were serine/threonine, we speculate that the MUC3A gene may encode the relevant amino acids and then play an important role in the pathogenesis of IMN through the mannose-binding lectin pathway. Previous evidence suggests that PLA2R-IgG4 can play a role by activating the complement lectin pathway with MBL [29]. The serine of the MUC3A gene also plays a role in the lectin binding pathway. Therefore, we further speculated that the MUC3A gene may be associated with IgG4 and anti-PLA2R antibody expression. There were some relationships in the diagnosis and prognosis of IMN. In addition, it was reported [30] that MUC3A is a class of membrane-associated mucins, which can mediate some of the particles and related pathogens adhering to the mucosal surface. Additionally, MUC3A is involved in binding of the receptor and ligand and signal transduction pathways. MUC3A can mediate the adhesion of the relevant particles to the membrane surface and participate in the receptor ligand binding process, suggesting that MUC3A may also play a role in the formation of immune complexes.

In addition, in this experiment, the genes for which we observed a significant difference in the circRNAs are mainly intron-derived circRNAs. The corresponding genes are SNORA25, SNORA31, SNORA70, SNORA75, SNORD112 and other small nucleolar RNAs (snoRNAs). An increasing number of studies have shown that snoRNAs can be further processed to form shorter RNA fragments, and these short fragments of snoRNAs have microRNA-like functions. This finding suggested that snoRNAs may act as microRNA precursors [31]. One study [32, 33] showed that circRNAs of different gene sources exist in different parts of the cell and the function is also different. The corresponding genes of circRNAs that we obtained in this experiment were mainly the intron source for coding snoRNAs. Therefore, we speculate that in the pathogenesis of IMN at the gene level, the circRNAs of the intron source may code snoRNAs that modify the mRNA during and before transcription as well as regulating the gene expression at the mRNA level.

Studies have shown that alleles-PLA2R1 and HLA-DQA1 are closely related to IMN [34]. In this study, we selected 29 metabolic pathways in the serum sample; 21 had PLA abnormalities. IMN does not appear to occur through a specific signaling pathway; instead, several pathways appear to work at the same time. Additionally, the corresponding gene of PLA was PLA2G4B, which corresponds to PLA2R positivity in IMN patients. IMN may be associated with the PLA2G4B gene. Therefore, evaluation of PLA2G4B may provide new clues for the diagnosis and treatment of IMN.

In conclusion, we found that there were abnormal expression levels of circRNAs in serum and urine exosomes in IMN patients. These circRNAs with abnormal expression could be involved in IMN pathogenesis. However, the specific mechanism and function of the circRNAs with differential expression in the disease require more direct evidence. However, with the continuous development of biological technology and continuous research on circRNAs, circRNAs will eventually provide a new theoretical basis in the disease diagnosis, treatment and prognosis. Additionally, the study of PLA2G4B may provide new clues for the diagnosis and treatment of IMN.

Acknowledgments

This study was financially supported by the Shenzhen Science and Technology Innovation Committee (grant no. JCYJ20160422151707152). This article used an English Language Service by American Journal Experts.

Hualin Ma and Ying Xu contributed equally to the work.

Conflict of interest

The authors declare no conflict of interest.

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