Roles and functions of exosomal miRNA in abdominal aortic aneurysm

Background and objective: abdominal aortic aneurysms (cid:0) AAAs (cid:0) are the permanent dilatation of the abdominal aorta, ruptured AAA is a serious threat to the patient's life. It’s hardly known about exosomal miRNAs in AAA. The main purpose of this article is to screen miRNAs which differentially expressed in exosomes from normal people and patients with AAA, and to understand the mechanism of work. Material and methods: The plasma of healthy control group and patients with AAA were collected, and the RNAs from exosomes was isolated and sequenced. The mature miRNA sequence of miRBase 21 database was used to identify the known type and expression of miRNAs. DEGseq software was used to analyze the types of miRNAs with signicant difference between the experimental group and the control group ( P (cid:0) 0.05, | log 2 (FoldChange) | ≥ 1 ). The targets of miRNAs were detected by miRTarBase, miRDB, TargetScan and miRWalk software. Targets were analyzed based on Kyoto encyclopedia of genes and genomes (cid:0) KEGG (cid:0) biological pathway and gene ontology (cid:0) GO (cid:0) functional enrichment analysis. Conclusion: miRNAs in exosomes regulate in the progress of AAA by activating PI3K-Akt/mTOR and MAPK pathway, and its mechanism needs more research. Background and objective: abdominal aortic aneurysms (cid:0) AAAs (cid:0) are the permanent dilatation of the abdominal aorta, ruptured AAA is a serious threat to the patient's life. It’s hardly known about exosomal miRNAs in AAA. The main purpose of this article is to screen miRNAs which differentially expressed in exosomes from normal people and patients with AAA, and to understand the mechanism of work. Material and methods: The plasma of healthy control group and patients with AAA were collected, and the RNAs from exosomes was isolated and sequenced. The mature miRNA sequence of miRBase 21 database was used to identify the known type and expression of miRNAs. DEGseq software was used to analyze the types of miRNAs with signicant difference between the experimental group and the control group ( P (cid:0) 0.05, | log 2 (FoldChange) | ≥ 1 ). The targets of miRNAs were detected by miRTarBase, miRDB, TargetScan and miRWalk software. Targets were analyzed based on Kyoto encyclopedia of genes and genomes (cid:0) KEGG (cid:0) biological pathway and gene ontology (cid:0) GO (cid:0) functional enrichment analysis. Conclusion: miRNAs in exosomes regulate in the progress of AAA by activating PI3K-Akt/mTOR and MAPK pathway, and its mechanism needs more research.

Introduction AAA refers to the abdominal aortic dilatation with a diameter of 3.0 cm or more. Most AAA are asymptomatic before rupture. Although the risk of rupture varies greatly, it depends on the size of the aneurysm. The risk of death associated with rupture is as high as 81%. The prevalence of AAA is 4-7% in men and 1-2% in women over 65 years old. At present, the treatment options for the prevention of aortic rupture are limited to surgical repair, and there is ine cient drug treatment to prevent progressive growth or rupture. The pathological features of AAA include vascular endothelial cell damage and in ammatory cell in ltration, extracellular matrix loss and vascular smooth muscle cell (VSMC) apoptosis. miRNA guides the RNA-induced silencing complex(RISC)to degrade mRNA or hinder its translation by pairing with the target gene mRNAs. miRNAs are closely related to a variety of diseases, including cancer, vascular diseases, immune system diseases so on. Studying the difference of miRNA expression in healthy and diseased state can be used to detect and monitor the disease process, and may become a biomarker of early diagnosis and prognosis of the disease, and look for potential regulatory mechanisms through its target genes.Therefore, exosomal miRNAs may be a target for early screening and treatment of AAA.
Material And Methods 1.1 Sample collection: Plasma samples from 3 patients with AAA and 3 healthy people were provided by the First A liated Hospital of Zhengzhou University. All patients with AAA were con rmed by CTA that the diameter of the abdominal aorta was larger than 3 cm (Table 1). healthy group without AAA was selected as the control group. The blood is collected into the EDTA anticoagulant tube and gently mixed to ensure exposure to the wall of the tube coated with EDTA. 1.2 Exosome separation: Firstly, the plasma sample was centrifuged at 2000 g at room temperature for 20 min, the residual cells and fragments were removed. Then, the supernatant was absorbed into the new tube, centrifuged at 10000 g at room temperature for 20 min again. According to the agreement, the supernatant was fully mixed with 1/3 volume Ribo Exosome Isolation Reagent RIBOBIO China standing at 4 ℃ for 30 min, followed by 15000 g centrifugation for 2 min to remove the supernatant. Phosphate buffered saline was used to re-suspend exosome particles.
1.3 Exosome identi cation: Using Zetasizer Nano-ZS (Malvern Panalytical, UK), the molecular diameter distribution determined by Nanoparticle Tracking Analysis (NTA) software is mainly composed of 20-200 nm particles. Fluorescent direct labeling CD63 and CD81 antibody (BD, USA) were used for staining, and unstained exosome was labeled as NC as negative control. Accuri C6 ow cytomenter (BD, USA ) was used for ow cytometry analysis ( 83%), which proved that the isolated EV contained abundant exosomes. The results are shown in Fig 1. Characterization of EV particle diameter is shown in Table 2.

Analysis of high-throughput sequencing data
The clean reads was obtained by removing the joint sequence, length 17 nt and low quality reads. Clean reads were compared with human genome by Burrow-Wheeler Aligner software. Comparing and annotating clean data with a variety of RNA databases 11 . Using Rfam 11.0 for yRNA, rRNA, snRNA, snoRNA and tRNA. MiRBase 21 for miRNA, piRNABank for piRNA 12 .
Compared with the entire reference sequence of miRBase 21, the number of miRNA expressions in each sample was obtained and standardized as number of mapped reads per million clean reads (RPM) 13 . The analysis of differential miRNA expression is an independent hypothesis statistical test for thousands of miRNAs. This multiple test has the problem of high false positive. In order to highlight the difference in expression, the P value needs to be corrected. We use DEGseq to correct the P value to get the Q value.
The lower the Q value is, the more signi cant the difference in miRNA expression is 14  Targets of miRNA with signi cant differences were predicted by TargetScan, miRDB, miRTarBase and miRWalk software, and the intersections were selected 15,16 . The targets were annotated in the KEGG biological pathway database, and the biological pathway enrichment analysis of targets was carried out by using Fisher Exact Test, with the threshold of P 0.05 17 . According to GO gene annotation, all genes of this species were selected as background genes, and hypergeometric method was used to calculate high frequency annotation(P 0.05) 18 .

Results
DEGseq helps us to screen out 85 species of miRNA with signi cant differences in expression, including 42 up and 43 down Table 3 . In order to study the potential biological function and pathway of miRNA, the predicted targets were used to analyze the enrichment of GO biological process and KEGG pathway.  are recommended for regular monitoring , . However, there is no reliable drugs that can limit the growth and rupture of AAA. Therefore, it is necessary to determine the pathways that can easily lead to the formation of aneurysms, which is very important for the discovery of new therapeutic targets.
Exosomes are the carrier of intercellular information. Proteins, lipids and RNA are transported from exosomes into receptor cells, effectively altering their biological responses. Our previous research proved that exosomal AFAP1-AS1 induces drug resistance of trastuzumab by binding to AUF1. Exosomal miRNA is stable and convenient for body uid detection, so it can be used as a diagnostic marker. Such as exosomal miR-22-3p and miR-320a were signi cantly increased in the patients with endometriosis, the two miRNAs may be potential diagnostic biomarkers for endometriosis. In addition, the exosomes' ability to transport functional vesicles to diseased cells also makes it a therapeutic carrier. Combining with new ligands, exosomes are expected to become the next generation of nanocapsules for precision medicine.
Exosome plays an important role in the progression of AAA. Recent study has shown that macrophagederived exosomes prompt the expression of metalloproteinases (MMP-2) in VSMC to regulate the progression of AAA through Jun N-terminal kinase (JNK) and P38 pathways. However, it's little known about the role of exosomal miRNA in AAA. By comparing the expression of exosomal miRNA in plasma samples of healthy people and AAA population, we found that there were 85 signi cantly different miRNAs, suggesting that miRNAs in exosomes work in the development of AAA. We sorted out these miRNAs and found that the mechanisms of some miRNAs in AAA have been reported. Down-regulated miR-145 affect the progression of AAA by regulating the target gene Rac2, which prevents atherosclerotic plaque calci cation by inhibiting the expression of Rac1-dependent macrophage interleukin-1β. Most AAA was considered to be one of the terminal manifestations of atherosclerosis , . Gan et al. reported that the overexpression of miR-15b reduces ACSS2 acts on AAA through lipid biosynthesis and in ammatory response. Up-regulation of miR-15a-5p is involved in the AAA by affecting the expression of cyclindependent kinase inhibitor 2B (CDKN2B), which was considered closely related to VSMC apoptosis. Down-regulated expression of miRNA-125b and miR-193b in AAA leads to the up-regulation of ALOX5 expression, which leads to the increase of leukotriene production and promote in ammation and injury of aortic wall. The overexpression of miR-29c-3p inhibits aortic brosis through targeting transforming growth factor β2(TGFβ2)and MMP2, which has a protective effect on the occurrence of AAA. In a word, the above results not only present that the isolated miRNA is reliable, but also prove that exosomal miRNA is crucial in the AAA. However, because a single mRNA is regulated by a variety of miRNAs, its mechanism is complex. As diagnostic marker of AAA, exosomoal miRNA needs more research to identify.
Interestingly, our study found that hsa-let-7i-5p was down-regulated in the AAA group, while hsa-let-7d-3p in the same family was up-regulated. Ana et al. reported that hsa-let-7i-5p regulate β-amyloid peptides by targeting metallopeptidase domain 10, amyloid protein-binding protein 2 and β-amyloid precursor. Zhang et al. found that hsa-let-7i-5p and its target gene Was1 mediate macrophage phagocytosis. A recent study con rmed that hsa-let-7i-5p is associated with endothelial progenitor cell dysfunction. It is hardly known about the role of hsa-let-7d-3p, and a study suggested that it may be associated with primary Sjogren's syndrome. Structurally, seed regions are based on the 2-8 nucleotide sequence at the 5 'end, which identify the 3' untranslated regions (UTR) of mRNA. The seed regions of the same family are highly conservative and the base difference is small. Studies showed that the complementary probability of miRNA 3' UTR and target genes is also the structural basis of different miRNA expression in the same family. It is an interesting phenomenon, showing that there is a competitive inhibition relationship. Besides, the mechanism of mir-193a and mir-125a is not understood, but the expression is also downregulated, so it may have similar effects to mir-193b and mir-125b.
In addition, some enriched signaling pathways have been con rmed to be related to vascular endothelial cell senescence and VSMC dysfunction. MTOR is a serine/threonine kinase that regulates cell growth and metabolism. Its function is realized by two complexes, mTORC1 and mTORC2. An article showed that mTORC1-S6K1-eNOS-uncoupling promotes the senescence of vascular endothelial cells. PI3K/Akt as the classic upstream pathway of mTOR, a study con rmed that PI3K/Akt-mTORC-S6K1 promotes VSMC apoptosis by regulating oxidative emergency response and telomere function . It is one of the pathological manifestations of AAA. Study also showed that AMPK may indirectly activate SIRT1 to inhibit mTORC1-S6K1 pathway, which may become a target to treat AAA. MAPK signaling pathway is involved in many important cellular reactions, and its activation leads to the phosphorylation of intracellular substrates or targets. There are 14 known MAPKs, including extracellular signal-regulated kinase(ERK1, ERK2, ERK3, ERK4, ERK5, ERK7), P38 α, β, γ, δ , JNK 1, 2, 3 , NLK. As mentioned earlier, the promotion of MMP-2 expression by JNK and P38 indicates the role of MAPK signaling pathway in AAA. A study proved that the activation of MAPK pathway regulates the contraction of angiotensin converting enzyme II to VSMCs, which eventually leads to VSMCs proliferation, differentiation and in ammation. Therefore, exosomal miRNAs may regulate the progression of AAA involved in VSMC through PI3K-Akt/mTOR and MAPK pathway. However, there are some shortcomings in our article, such as the insu cient number of samples, the action mechanism of most exosomal miRNAs is not understood. Further research is needed.

Conclusion
Our study shows that there is a difference in the expression of miRNA in exosomes between healthy people and AAA patients, and some known miRNAs have been con rmed to act on target genes in the form of exosome in peripheral blood, such as miR-145, miR-15b, miR-15a-5p, miR-29c-3p ,miR-193band miR-125b. These miRNAs may regulate PI3K-Akt/mTOR and MAPK pathway to cause vascular smooth muscle dysfunction and induce AAA.

Declarations
Authors' contributions CH and LZ designed the study and revised the manuscript. ZYB, LC and GYM performed the experiment.
WSW and CGP analyzed the data. JZY, HZH, XP and LSR collected the samples. ZYB and WSW were two major contributors in writing the manuscript. All authors read and approved the nal manuscript.
Funding Figure 1 Characterization of EVs from plasma samples of AAA patients and controls. A Size distribution of plasma EVs by NTA. B Plasma EVs were analyzed by ow cytometry for the exosomal markers antibodies CD63 and CD81.

Figure 2
Sequence length distribution analysis of EVs by RNA-Seq.

Figure 3
Volcanic diagram of miRNA differential expression analysis among samples. The horizontal line indicates P value of 0.05, the vertical lines correspond to 2-fold upregulation and downregulation.