Primary open-angle glaucoma (POAG) is tightly related with extracellular matrix (ECM) remodeling of human trabecular meshwork cells (HTMCs). Transforming growth factor-β2 (TGF-β2) can induce ECM remodeling. The aim of the study was to investigate the microRNAs (miRNAs) expression changes of extracellular vesicles (EVs) derived from HTMCs treated with TGF-β2. EVs were isolated from HTMCs supernatant cultured for 24 h with TGF-β2. The morphology of EVs pellets was examined by transmission electron microscopy. Nanoparticle tracking analysis used to demonstrate the particle size distribution. Total EVs RNAs were extracted for subsequent miRNA gene chip analysis to investigate differentially expressed miRNAs between the controls and treatment cells. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict potential target and validate possible functions of the miRNAs. There were 23 miRNAs upregulated and 3 miRNAs downregulated and 469,102, and 94 GO terms involved in biological processes, cellular components, and molecular function for the possible functions of the 26 miRNAs. These findings indicate that TGF-β2 may alter EVs miRNAs expression to participate in the pathogenesis of POAG. They may provide significant information for potential biomarkers for POAG diagnosis and treatment.
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References
1.
(2000). The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am J Ophthalmol, 130, 429–440.
2.
AgarwalP., DaherA.M., and AgarwalR. (2015). Aqueous humor TGF-beta2 levels in patients with open-angle glaucoma: a meta-analysis. Mol vision, 21, 612–620.
3.
BaileyJ.N., YaspanB.L., PasqualeL.R., HauserM.A., KangJ.H., LoomisS.J., et al. (2014). Hypothesis-independent pathway analysis implicates GABA and acetyl-CoA metabolism in primary open-angle glaucoma and normal-pressure glaucoma. Hum Genet, 133, 1319–1330.
4.
ChenJ.L., SuY., and WangF. (2016). [Role of microRNA in primary open-angle glaucoma]. [Zhonghua yan ke za zhi] Chin J Ophthalmol, 52, 476–480.
5.
DismukeW.M., ChallaP., NavarroI., StamerW.D., and LiuY. (2015). Human aqueous humor exosomes. Exp Eye Res, 132, 73–77.
6.
DismukeW.M., KlingebornM., and StamerW.D. (2016). Mechanism of fibronectin binding to human trabecular meshwork exosomes and its modulation by dexamethasone. PLoS One 11,e0165326.
7.
DrewryM.D., ChallaP., KuchteyJ.G., NavarroI., HelwaI., HuY., et al. (2018). Differentially expressed microRNAs in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma. Hum Mol Genet, 27, 1263–1275.
8.
GiroudM., KarbienerM., PisaniD.F., GhandourR.A., BerangerG.E., NiemiT., et al. (2016). Let-7i-5p represses brite adipocyte function in mice and humans. Sci Rep 6,28613.
9.
GottankaJ., ChanD., EichhornM., Lutjen-DrecollE., and EthierC.R. (2004). Effects of TGF-beta2 in perfused human eyes. Invest Ophthalmol Vis Sci, 45, 153–158.
10.
HardingC.V., HeuserJ.E., and StahlP.D. (2013). Exosomes: looking back three decades and into the future. J Cell Biol, 200, 367–371.
11.
HardyK.M., HoffmanE.A., GonzalezP., McKayB.S., and StamerW.D. (2005). Extracellular trafficking of myocilin in human trabecular meshwork cells. J Biol Chem, 280, 28917–28926.
12.
HindleA.G., ThoonenR., JasienJ.V., GrangeR.M.H., AminK., WiseJ., et al. (2019). Identification of Candidate miRNA Biomarkers for Glaucoma. Invest Ophthalmol Vis Sci, 60, 134–146.
13.
HoffmanE.A., PerkumasK.M., HighstromL.M., and StamerW.D. (2009). Regulation of myocilin-associated exosome release from human trabecular meshwork cells. Invest Ophthalmol Vis Sci, 50, 1313–1318.
14.
IbrahimS.A., HassanH., and GotteM. (2014). MicroRNA-dependent targeting of the extracellular matrix as a mechanism of regulating cell behavior. Biochim Biophys Acta, 1840, 2609–2620.
15.
InataniM., TaniharaH., KatsutaH., HonjoM., KidoN., and HondaY. (2001). Transforming growth factor-beta 2 levels in aqueous humor of glaucomatous eyes. Graefes Arch Clin Exp Ophthalmol, 239, 109–113.
16.
IrizarryR.A., HobbsB., CollinF., Beazer-BarclayY.D., AntonellisK.J., ScherfU., et al. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics, 4, 249–264.
17.
JayaramH., PhillipsJ.I., LozanoD.C., ChoeT.E., CepurnaW.O., JohnsonE.C., et al. (2017). Comparison of MicroRNA Expression in Aqueous Humor of Normal and Primary Open-Angle Glaucoma Patients Using PCR Arrays: a Pilot Study. Invest Ophthalmol Vis Sci, 58, 2884–2890.
18.
KaloucheG., BoucherC., CosteA., DebusscheL., OrsiniC., BaudouinC., et al. (2016). Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53. Biochim Biophys Acta, 1863, 2322–2332.
19.
KellerK.E., BhattacharyaS.K., BorrasT., BrunnerT.M., ChansangpetchS., ClarkA.F., et al. (2018). Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp Eye Res, 171, 164–173.
20.
KhushmanM., BhardwajA., PatelG.K., LauriniJ.A., RovedaK., TanM.C., et al. (2017). Exosomal Markers (CD63 and CD9) Expression pattern using immunohistochemistry in resected malignant and nonmalignant pancreatic specimens. Pancreas, 46, 782–788.
21.
KobayashiM., SalomonC., TapiaJ., IllanesS.E., MitchellM.D., and RiceG.E. (2014). Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200. J Transl Med 12,4.
22.
KogureT., LinW.L., YanI.K., BraconiC., and PatelT. (2011). Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth. Hepatology (Baltimore, Md), 54, 1237–1248.
23.
KwonH.S., and TomarevS.I. (2011). Myocilin, a glaucoma-associated protein, promotes cell migration through activation of integrin-focal adhesion kinase-serine/threonine kinase signaling pathway. J Cell Physiol, 226, 3392–3402.
24.
KwonY.H., FingertJ.H., KuehnM.H., and AlwardW.L. (2009). Primary open-angle glaucoma. N Engl J Med, 360, 1113–1124.
25.
LasserC., AlikhaniV.S., EkstromK., EldhM., ParedesP.T., BossiosA., et al. (2011). Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9,9.
26.
LernerN., AvissarS., and Beit-YannaiE. (2017). Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system. PLoS One 12,e0171153.
27.
LiM., ZeringerE., BartaT., SchagemanJ., ChengA., and VlassovA.V. (2014). Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc Lond B Biol Sci 369,20130502.
28.
LiuY., AllinghamR.R., QinX., LayfieldD., DellingerA.E., GibsonJ., et al. (2013). Gene expression profile in human trabecular meshwork from patients with primary open-angle glaucoma. Invest Ophthalmol Vis Sci, 54, 6382–6389.
29.
LiuY., BaileyJ.C., HelwaI., DismukeW.M., CaiJ., DrewryM., et al. (2016). A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD Consortium. Invest Ophthalmol Vis Sci, 57, 3974–3981.
30.
LunaC., LiG., HuangJ., QiuJ., WuJ., YuanF., et al. (2012). Regulation of trabecular meshwork cell contraction and intraocular pressure by miR-200c. PLoS One 7,e51688.
31.
LunaC., LiG., QiuJ., EpsteinD.L., and GonzalezP. (2009). Role of miR-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress. Mol Vis, 15, 2488–2497.
32.
LunaC., LiG., QiuJ., EpsteinD.L., and GonzalezP. (2011). Cross-talk between miR-29 and transforming growth factor-betas in trabecular meshwork cells. Invest Ophthalmol Vis Sci, 52, 3567–3572.
33.
MengB., LiH., SunX., QuW., YangB., ChengF., et al. (2017). sigma-1 receptor stimulation protects against pressure-induced damage through InsR-MAPK signaling in human trabecular meshwork cells. Mol Med Rep, 16, 617–624.
34.
Navarro-QuirozE., Pacheco-LugoL., Navarro-QuirozR., LorenziH., Espana-PucciniP., Diaz-OlmosY., et al. (2017). Profiling analysis of circulating microRNA in peripheral blood of patients with class IV lupus nephritis. PLoS One 12,e0187973.
35.
OverbyD.R., StamerW.D., and JohnsonM. (2009). The changing paradigm of outflow resistance generation: towards synergistic models of the JCT and inner wall endothelium. Exp Eye Res, 88, 656–670.
36.
PanC., StevicI., MullerV., NiQ., FerrerL.O., PantelK., et al. (2018). Exosomal microRNAs as tumor markers in epithelial ovarian cancer. Mol Oncol, 12, 1935–1948.
37.
PerkumasK.M., HoffmanE.A., McKayB.S., AllinghamR.R., and StamerW.D. (2007). Myocilin-associated exosomes in human ocular samples. Exp Eye Res, 84, 209–212.
38.
PolanskyJ.R., FaussD.J., and ZimmermanC.C. (2000). Regulation of TIGR/MYOC gene expression in human trabecular meshwork cells. Eye (Lond), 14 (Pt 3B), 503–514.
ShepardA.R., MillarJ.C., PangI.H., JacobsonN., WangW.H., and ClarkA.F. (2010). Adenoviral gene transfer of active human transforming growth factor-{beta}2 elevates intraocular pressure and reduces outflow facility in rodent eyes. Invest Ophthalmol Vis Sci, 51, 2067–2076.
41.
SkarieJ.M., and LinkB.A. (2008). The primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway. Hum Mol Genet, 17, 2474–2485.
42.
StamerW.D., HoffmanE.A., LutherJ.M., HacheyD.L., and ScheyK.L. (2011). Protein profile of exosomes from trabecular meshwork cells. J Proteomics, 74, 796–804.
43.
TabakS., Schreiber-AvissarS., and Beit-YannaiE. (2018). Extracellular vesicles have variable dose-dependent effects on cultured draining cells in the eye. J Cell Mol Med, 22, 1992–2000.
44.
TheryC., AmigorenaS., RaposoG., and ClaytonA. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3, Unit 3.22.
45.
TkachM., and TheryC. (2016). Communication by extracellular vesicles: where we are and where we need to go. Cell, 164, 1226–1232.
46.
Tovar-VidalesT., ClarkA.F., and WordingerR.J. (2011). Transforming growth factor-beta2 utilizes the canonical Smad-signaling pathway to regulate tissue transglutaminase expression in human trabecular meshwork cells. Exp Eye Res, 93, 442–451.
47.
UsubaW., UrabeF., YamamotoY., MatsuzakiJ., SasakiH., IchikawaM., et al. (2018). Circulating miRNA panels for specific and early detection in bladder cancer. Cancer Sci, 110, 408–419.
48.
VillarrealG.Jr., ChatterjeeA., OhS.S., OhD.J., KangM.H., and RheeD.J. (2014). Canonical wnt signaling regulates extracellular matrix expression in the trabecular meshwork. Invest Ophthalmol Vis Sci, 55, 7433–7440.
49.
VillarrealG.Jr., OhD.J., KangM.H., and RheeD.J. (2011). Coordinated regulation of extracellular matrix synthesis by the microRNA-29 family in the trabecular meshwork. Invest Ophthalmol Vis Sci, 52, 3391–3397.
50.
WangW., GaoJ., and WangF. (2017a). MiR-663a/MiR-423-5p are involved in the pathogenesis of lupus nephritis via modulating the activation of NF-kappaB by targeting TNIP2. Am J Transl Res, 9, 3796–3803.
51.
WangY., LiF., and WangS. (2016). MicroRNA93 is overexpressed and induces apoptosis in glaucoma trabecular meshwork cells. Mol Med Rep, 14, 5746–5750.
52.
WangY., ZhangG., KangL., and GuanH. (2017b). Expression profiling of DNA methylation and transcriptional repression associated genes in lens epithelium cells of age-related cataract. Cell Mol Neurobiol, 37, 537–543.
53.
YangX., LiuB., BaiY., ChenM., LiY., ChenM., et al. (2011). Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells. Mol Vis, 17, 2978–2985.
54.
YooJ.K., KimJ., ChoiS.J., NohH.M., KwonY.D., YooH., et al. (2012). Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells. Stem Cells Dev, 21, 2049–2057.
55.
ZhangC., ChenB., JiaoA., LiF., SunN., ZhangG., et al. (2018). miR-663a inhibits tumor growth and invasion by regulating TGF-beta1 in hepatocellular carcinoma. BMC Cancer 18,1179.
56.
ZhouF., YuanQ., ZhangW., NiuM., FuH., QiuQ., et al. (2018a). MiR-663a stimulates proliferation and suppresses early apoptosis of human spermatogonial stem cells by targeting NFIX and regulating cell cycle. Mol Ther Nucleic Acids, 12, 319–336.
57.
ZhouX., LuZ., WangT., HuangZ., ZhuW., and MiaoY. (2018b). Plasma miRNAs in diagnosis and prognosis of pancreatic cancer: a miRNA expression analysis. Gene, 673, 181–193.
58.
Zlotogorski-HurvitzA., DayanD., ChaushuG., SaloT., and VeredM. (2016). Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals. J Cancer Res Clin Oncol, 142, 101–110.
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