Altered expression of exosomal miRNAs from primary human trabecular meshwork cells induced by transforming growth factor-β2

Abstract


Background
Glaucoma is an irreversible ocular disease and the main cause of blindness around the world.It is characterized by optic neuropathy and corresponding visual eld loss [1].The most common form of glaucoma, primary open-angle glaucoma(POAG), is estimated to affect 58.6 million individuals by 2020 [2].Elevated intraocular pressure (IOP) is the only modi able risk factor for POAG, presumably due to increased resistance in the conventional out ow pathway including trabecular meshwork (TM) and Schlemm's canal [3,4].The pathophysiological processes leading to increased resistance in out ow pathways is yet to be fully elucidated.However, increased out ow resistance has been strongly correlated with aberrantly elevated levels of TGF-β2 within the aqueous humor (AH) of POAG patients [5,6].
Experiments in vitro and in vivo have shown that TGF-β2 can enhance the synthesis and deposition of extracellular matrix (ECM) components; also, it inhibits TM cell proliferation and increases TM cell phagocytosis.Therefore, TGF-β2 might play a vital role in the pathogenesis in POAG [7][8][9].In this process, many genes altered expression is related to the ECM remodeling, but the exact regulation mechanism is unclear.Other researches have shown that TGF-β2 could control miRNAs expression, which affected the metabolism of ECM in the TM [10][11][12].
Many miRNAs were found in TM tissue and AH.Some miRNAs expression may affect the TM function and the synthesis of ECM in different ways [13][14][15].For example, miR-29 family functions as a critical suppressor of various ECM proteins in the TM and can be modi ed by TGF-β2 [12].Some miRNAs maybe maintain homeostasis of IOP by regulating the target genes in TM [15][16][17].However, the mechanism behind changes of miRNAs is unrevealed, and it is not clear how the miRNAs regulate gene expression or affect ECM function.
MiRNAs are found not only within but also outside of the cell, i.e., in the form of the exosome.Recent studies have demonstrated that exosomes may be involved in the regulation of IOP [18][19][20][21][22]. Exosomes are nanovesicles (30-150 nm in diameter).They are released into the extracellular environment by nearly all cells of the human body under both physiological and pathological conditions, including intraocular tissue cells [23,24] and were detected in a number of body uids, including AH, blood plasma, urine, saliva and breast milk [20,[25][26][27].Exosomes could carry coding and non-coding RNA, single strand DNA, lipids, proteins and antigen-presenting molecules [28].Exosomes are known for its function in antigen presentation and cell-cell communication.Consequently, they are recognized as important biomarkers of human disease [29].The glaucoma-causing protein, myocilin, is an intracellular protein and released from TM cells by exosomes [18,22,30].A previous study showed the physical properties of AH exosomes and pointed out that the exosomes in AH contain characteristic miRNAs [20].
In recently years, there is growing interest in studying the exosomal miRNAs for POAG.We still lack an understanding whether TGF-β2 treatment TM cells, a model of glaucomatous TM dysfunction, can produce changes in exosomal cargo and function especially for exosomal miRNAs.The overall goal of this study was to identify and characterize exosomal miRNAs expression pro les in human trabecular meshwork cells (HTMCs) treatment by TGF-β2 and speculate their potential target via bioinformatics analysis.The nding could be further developed as therapeutics for the POAG diagnosis and treatment.

Conditioned medium collection and exosome isolation
To better understand the role of exosomes in HTM biology, our rst step was to purify exosomes from conditioned media of primary HTMCs monolayers.After 24 h treatment, the cells culture supernatant was collected and exosomes were isolated by differential ultracentrifugation as previously described [33].Brie y, the cells culture supernatant was centrifuged for 5 min at 300 g and then 10 min at 2000 g to eliminate cellular contamination.Then, the supernatant was centrifuged at 10000 g for 30 min and ltered by a 0.22 µm lter (Millipore, USA), followed by ultracentrifugation at 100000 g for 1 h to pellet the exosomes.The pellets were washed in 20 ml cold PBS and pelleted again at 100000 g for 1 h, and resuspended in sterile PBS.All the operations were performed at 4 °C.The extracted exosomes were allocated and stored at -80 °C or used for the downstream experiments.The ultrastructure of exosomes was examined by transmission electron microscope (TEM) as previously described [33].For the purpose of demonstrating the particle size distribution, nanoparticle tracking analysis (NTA) was performed using NanoSight NS300 (Malvern Instruments, Inc., Westborough, MA, USA) according to the operating instructions.Exosomal membranes were enriched in endosome-speci c tetraspanins [34].Then, we used Western Blot to detect the expression of the common exosomal markers CD63, CD81 and CD9 as previously described [35].

Total exosomal miRNAs extraction and analysis
To determine the expression changes of exosomal miRNAs derived from HTMCs when they were treated with TGF-β2, we used miRNA gene chip to analysis.First, miRNAs were extracted from exosomes using TRIzol reagent (Invitrogen; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) as previously described [36].
A miRNA library preparation was conducted under the assistance of Shanghai Genechem Co., Ltd.The quality and concentration of miRNAs were assessed using NanoDrop 2000 Spectrophotometer (NanoDrop Technologies; Thermo Fisher Scienti c, Inc.).MiRNAs content were assessed using an Agilent 2200 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA).Total miRNAs were labeled using FlashTag Biotin HSR labeling kits (Affymetrix, CA, USA) and hybridized to Affymetrix Gene Chip miRNA 4.0 Arrays, and scanned using an Affymetrix GCS3000 Gene Array Scanner according to manufacturer's protocol (Affymetrix, Santa Clara, CA, USA).

Bioinformatics analysis
The comprehensive function annotations of the potential targets of exosomal miRNAs were performed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis based on the DAVID 6.7 software (http://david.abcc.ncifcrf.gov/home.jsp).GO analysis was applied to predict the main functions of the target genes according to the GO project.Pathway analysis was performed to determine the signi cant pathway of the differential genes according to the KEGG.MiRNA-target gene network was established on the basis of GO and KEGG predicted data for the illustration of the relationship between miRNAs and their target genes.The target genes of exosomal miRNAs were predicted using StarBase (http://starBase.sysu.edu.cn/).

Statistical analysis
Fisher's exact test and χ2 were used to classify the GO category and select the signi cant pathway.All GO terms and signaling pathways were analyzed with a threshold of signi cance that was de ned by a Pvalue of < 0.05.Unless otherwise noted, all results were obtained through a minimum of three independent experimental replications.

Human trabecular meshwork cell released exosomes
Like most other cells, cultured HTMCs can also release extracellular vesicles with properties consistent with exosomes [21].After isolation, the morphology of exosome pellets was examined by TEM revealing membrane-bound particles that were homogeneous in appearance (Fig. 1A).In addition, the size distribution of exosome pellets was analyzed using NTA and the results revealed prospective diameters which ranged from 30-120 nm (Fig. 1B).Furthermore, we detected three well-established surface markers CD63, CD81 and CD9 for exosomes (Fig. 1C).

Exosomal miRNA-miRNA co-expression network
To gain a better understanding of how these miRNAs relate to one another, we then constructed the coexpression network of exosomal miRNAs (Fig. 3).In total, except hsa-miR-6724-5p, hsa-miR-6800-3p and hsa-miR-6716-3p, the other 23 miRNAs were included in the interaction network.In the interaction network, many up-regulated miRNAs have a positive correlation with another miRNA.Hsa-miR-1915-3p, hsa-miR-6727-5p and hsa-miR-3960 displayed the highest co-expression degree compared with other miRNAs.Has-let-7i-5p, which was down-regulated in exosome had a negative correlation with miR-4058.However, hsa-miR-6800-3p and hsa-miR-6716-3p weren't in this network.The co-expressed interaction network indicated that there may be synergistic and antagonistic effects between miRNAs.One single miRNA might interact with other several miRNAs and vice versa.

GO and KEGG pathway enrichment analysis
To interpret possible physiological or pathological processes and pathways regulated by the identi ed miRNAs, their putative target genes were subjected to GO annotation and KEGG pathway analysis (Fig. 4A, B).The results revealed that a total of 469 GO terms were involved in biological processes with P < 0.05 (The top 3: antigen processing and presentation, regulation of microtubule cytoskeleton organization and regulation of transcription from RNA polymerase II promoter in response to stress).Up to 102 GO terms were in relation to cellular components with P < 0.05 (The top 3: spliceosomal complex, endoplasmic reticulum lumen and neuron projection cytoplasm).There were 94 GO terms related to molecular function with P < 0.05 (The top 3: insulin-like growth factor I binding, nucleoside-triphosphatase regulator activity and transferase activity, transferring acyl groups).To provide information about the possible functions of 26 differentially expressed miRNAs, target prediction analysis was conducted by StarBase.The top 5 pathways, with the lowest P-value, were the p13k-akt signaling pathway, human papillomavirus infection, human cytomegalovirus infection, MAPK signaling pathways and proteoglycans in cancer.These results indicated that exosomal miRNAs involve a variety of cellular physiological and pathological processes.
Selective signaling pathways-exosomal miRNA coexpression network Many signaling pathways are involved in the physiological and pathological changes of TM cells, and thus participate in the pathogenesis of POAG, like p53 signaling pathway [37,38], focal adhesion [39,40], MAPK signaling pathway [41,42].We then constructed the co-expression network of selective signaling pathways and exosomal miRNAs (Fig. 5).This network indicated that one single miRNA was bound up with a couple of signaling pathways and vice versa.

Discussion
Previous studies suggested that elevated level of TGF-β2 in AH of POAG maybe central to the pathologic behavior of the disease [5,7,8].However, until now, a comprehensive investigation of the effects of TGF-β2 has not been presented.MicroRNAs housed within exosomes have emerged in recent years as critical modulators to synchronize AH production and drainage for ultimately IOP homeostasis [43] [20].Our study was designed to test whether a famous model of glaucomatous TM dysfunction, TGF-β2 treatment, can produce pathological changes in exosomal cargo and function.Furthermore, we want to reveal possible physiological or pathological processes and pathways involving pathogenesis of POAG regulated by the identi ed exosomal miRNAs.
According to the results of microarray analysis, 23 miRNAs were revealed to be up-regulated in the model group compared with the control group.Hsa-miR-6087, hsa-miR-663a and hsa-miR-6821-5p were the top three up-regulated exosomal miRNAs, whereas hsa-miR-6800-3p, hsa-miR-6716-3p, and hsa-let-7i-5p were revealed to be markedly down-regulated.Hsa-miR-6087 has been previously reported to act as an important mediators of cancer [44,45],intermediate monocytes [46]and regulation of cell differentiation [47].Has-miR-663a is located in the chromosome 20q11.1 and has been reported to be closely related with the biological behavior of cell differentiation, in ammation, autoimmune diseases, and cancer [48][49][50].Let-7 was discovered early as a member of the large class of non-protein-coding RNAs.Dysregulated expression of the let-7i-5p is associated with various diseases such as cancer, and cardiovascular [51].These exosomal miRNAs of AH may play a crucial role in the cell-cell communication for pathological processes of TM from POAG patients.
Previous study showed that signi cant up-regulation of mir-518d and mir-143, and signi cant downregulation of mir-660 expression were observed in AH from POAG patients [52].For this phenomenon, we infer the miRNAs in AH are not parallel to the miRNAs of the exosomes.To date, the speci c source of miRNAs in AH is unknown.Variable intraocular cells can secrete exosomes into the AH, and numerous molecules, particularly miRNAs, have been shown to change in response to ocular disease or injury.
Recent study shows that non-pigmented ciliary epithelium cell (NPCE)-derived exosomes could affect canonical Wnt signaling in TM cells, a pathway involved in IOP regulation [53].Thus, we suspect that the changes of exosomal miRNAs mediate cell-cell communication and play a major role in the crosstalk between TM cells and the macro−/microenvironment.
The miRNA-miRNA interaction network showed that during the occurrence and development of diseases.Many miRNA molecules are involved in the process.The term interaction means one miRNA may interact and regulate the stability or expression of the other one or the others.Each miRNA regulates a broad spectrum of target genes and affects many signaling pathways.Therefore, disruption of a single miRNA expression may only limit an inhibitory effect, whereas joint interference of multiple miRNAs may be effective.Thus, determining how to silence multiple abnormally expressed miRNAs simultaneously in order to enhance the e cacy of disease treatments is important.For the resaon, we used DIANA-TarBase to predicte the potential targets of 26 exosomal miRNAs.Unlike miRDB and TargetScan, DIANA-TarBase was the rst database aiming to catalog published experimentally validated interactions between miRNAs and genes.The GO analysis results revealed that the exosomal miRNAs were involved in biological processes, cellular components and molecular functions, including antigen processing and presentation, spliceosomal complex and insulin-like growth factor I binding.These miRNAs are not speci c to a single gene.These miRNAs alter the expression of a number of genes instead of a single gene, and these genes may be involved in a single cellular process like extracellular matrix remodeling [54].
KEGG pathway enrichment analysis also revealed that the p13k-akt signaling pathway and MAPK signaling pathways may be involved in the process.We then constructed the co-expression network of selective signaling pathways and exosomal miRNAs.Overall, these studies indicate that exosomes and their cargoes can be used as biomarkers for glaucoma, potentially serving as an early diagnostic marker, and may also contribute directly to the progression of the disease.TGF-β2 cause not only gene and protein expression changes in the HTM cells, but also can alter exosomal miRNAs expression.These miRNAs enlisted in the current study represent only a small proportion of genes in exosomes.This condition may be correlated with the speci c condition of in vitro culture of the cells.

Conclusions
The present study provided new information regarding the potential role of miRNAs in exosomes.MicroRNAs in AH exosome can be stably stored under different conditions, indicating that exosomal miRNAs are potential biomarkers or novel therapeutic tools of POAG.

Declarations
Ethics approval and consent to participate

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Figure 1 The
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Table 1
The change more than 2-fold for exosomal miRNAs of miRNA microarray analysis