miR-486-5p Restrains Extracellular Matrix Production and Oxidative Damage in Human Trabecular Meshwork Cells by Targeting TGF-β/SMAD2 Pathway

Background Glaucoma is characterized by elevated intraocular pressure caused by aqueous outflow dysfunction. Trabecular meshwork plays a key role in controlling intraocular pressure by modulating aqueous outflow. This study investigated the protective effects of miR-486-5p in H2O2-stimulated human trabecular meshwork cells (TMCs). Methods TMCs were disposed with 300 μM H2O2 to establish oxidative damage models in vitro. miR-486-5p mimics and its controls were transfected into TMCs, and cell apoptosis and extracellular matrix production (ECM) genes were measured by flow cytometry, western blotting, and immunofluorescence staining. Activities of superoxide dismutase (SOD) and malondialdehyde (MDA) were also assayed. Online tools and luciferase reporter assays were used to uncover the relationship between miR-486-5p and the TGF-β/SMAD2 pathway. Results We found that H2O2-induced oxidative damage in TMCs and miR-486-5p was downregulated in H2O2-stimulated TMCs. Overexpression of miR-486-5p mitigated H2O2-induced oxidative damage by inhibiting apoptosis, reducing cleaved caspase-3/9 expression, reducing MDA levels, and increasing SOD levels as well as downregulating ECM genes. SMAD2 was demonstrated to be targeted by miR-486-5p, and miR-486-5p inhibited TGF-β/SMAD2 signaling in H2O2-stimulated TMCs. Additionally, SMAD2 was upregulated by H2O2, and SMAD2 upregulation abrogated the protective effects of miR-486-5p against H2O2-induced injury. Conclusion miR-486-5p restrains H2O2-induced oxidative damage in TMCs by targeting the TGF-β/SMAD2 pathway.


Introduction
Glaucoma is a complex neurodegenerative disorder characterized by progressive optic neuropathy, which can result in blurred vision and even blindness [1]. e main risk factors of glaucoma include elevated intraocular pressure and insufficient blood supply to the optic nerve [2]. Currently, lowering intraocular pressure is the mainstream treatment in retarding the process of glaucomatous neuropathy, which can be achieved by medication, conventional surgery, and laser [3]. Although these remedies may rescue remaining eyesight, many patients fail to reach intraocular pressure targets [4]. Elevated intraocular pressure is a direct consequence of increased resistance to aqueous humor outflow [5]. It has been recognized that the trabecular meshwork (TM) exerts the main function on aqueous humor circulation [6,7]. Studies show that an extraordinary increase of extracellular matrix (ECM) molecules such as collagen and fibronectin in the TM is the main cause of outflow resistance [8,9]. erefore, there is an unmet need to elucidate the pathological mechanism related to excessive production of ECM from glaucomatous TM. e transforming growth factor-β (TGF-β) family modulates a range of cellular processes, including ECM synthesis, cell proliferation, and differentiation [10,11]. It is also recognized as a crucial profibrotic mediator in fibrotic disease. Studies show that the TGF-β/SMAD signaling pathway exerts key effects on regulating ECM synthesis in fibrotic processes [12]. In glaucomatous optic neuropathy, the TGF-β/SMAD pathway regulates key ECM genes, such as collagen, laminin, and fibronectin in the TM, which is associated with elevated intraocular pressure [13][14][15]. Previous reports indicated that TGF-β2-subjected human TMCs elicits a significant increase in SMAD2/3 signaling as well as an increase in collagen protein content [16]. Downregulation of SMAD7 interrupts the effects of TGF-β2 on the expression of several ECM components, and SMAD7 is a key molecular switch to inhibit TGF-β2 signaling in the TM [17,18]. Additionally, nonpigmented ciliary epithelium-derived extracellular vesicles loaded with SMAD7 siRNA attenuate Wnt signaling in TM cells, which may be beneficial as a therapeutic target to lower intraocular pressure [19]. erefore, it is needed to discover potential targets in the TGF-β/SMAD pathway to restrain ECM production in the TM.
MicroRNAs (miRNAs) contain approximately 18-22 nucleotides and participate in the regulation of the biological process by controlling gene expression post-transcriptionally [20]. In glaucoma, differentially expressed miRNAs in the aqueous humor and blood of patients have been identified and compared to matched control individuals [21,22]. Increasing studies have focused on the roles of miRNAs in modulating cellular function of the TM under various pathological conditions. Zhang et al. demonstrated that miR-181a improves the survival of TMCs under oxidative stress by blocking NF-κB signaling [23]. Overexpression of miR-183 reinforces DNA damage and promotes cellular senescence in the TM after ultraviolet irradiation by targeting KIAA0101 [24]. Additionally, miRNAs were proven to exert key functions in maintaining ECM homeostasis [25,26]. For example, miR-137 facilitates cell proliferation and downregulates ECM protein expression levels in H 2 O 2 -stimulated human TMCs through the YAP/TAZ pathway [27]. miR-483-3p attenuates ECM production in TMCs by targeting SMAD4 [28].
us, a future study is needed to explore whether restoration of miR-486-5p expression could block SMAD2 signaling in the TMCs, blocking ECM remodeling under mechanical stress.

Flow Cytometry for Transfection Efficiency Detection.
e transfected cells were collected and treated with 0.25% trypsin followed by resuspension in PBS. Cells were prepared into single-cell suspension which was then centrifuged at 800 rpm for 5 min. After that, the supernatant was removed, and cells were washed with PBS. Cells (1 × 10 6 cells/mL) were then subjected to transfection efficiency detection by a FACScan flow cytometer (Becton-Dickinson, CA, USA).

MTT Assay.
After treatment with concentrations of H 2 O 2 , TMCs were seeded in 96-well plates at 3 × 10 3 cells/ well in a 37°C incubator. Next, 20 μL MTT solution (5 mg/mL; Sigma-Aldrich) was added and incubated for 4 h at 37°C. e medium was removed, and the residue was dissolved with 100 μL dimethyl sulfoxide for 15 min. e optical density at 490 nm was measured with a SpectraMax M2e microplate reader (Molecular Devices, CA, USA).

Flow Cytometry.
Annexin V-FITC/PI Apoptosis Kits (cat. no. 70-AP101-100; Hangzhou, China) was utilized to analyze apoptosis. After treatment, TMCs were washed with cold PBS and suspended in 1× binding buffer (Sigma-Aldrich). Subsequently, 500 μL cell suspension (1 × 10 6 cell/mL) was added to a flow tube. Cells were then stained with 5 μL Annexin V-FITC and 10 μL PI, respectively, for 15 min in the dark. Finally, the stained cells were analyzed with a FACScan flow cytometer (Becton-Dickinson, CA, USA) and FlowJo software version 7.6.1.

ELISA.
e levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the cell medium were analyzed with SOD assay kits (cat. no. ab65354) and MDA assay kits (cat. no. ab118970) as per the manufacturer's protocols.
2.11. Luciferase Reporter Assay. HEK293Tcells or TMCs were seeded in 24-well plates. Luciferase reporter plasmids were constructed: pmirGLO-SMAD2-Wt containing the SMAD2 3'-UTR in the binding site for miR-486-5p and pmirGLO-SMAD2-Mut with a mutation at the predicted miR-486-5p binding site. After being cultured to 80% confluency, cells were transfected with 800 ng SMAD2-Wt or SMAD2-Mut plasmids, together with 20 nmol miR-486-5p mimics or NC mimics. After 48 h post-transfection, the activities of Renilla luciferase that were normalized to firefly luciferase activities were measured using a dual-luciferase reporter system (Promega).

Statistical Analysis.
All tests were conducted in at least 3 biological replicates. Comparisons were performed using unpaired Student's t-test and one-way ANOVA. Data were processed with SPSS v. 23.0 (IBM Corp., NY, USA) and are expressed as the mean ± SD. Statistical significance was set at p < 0.05.

Journal of Ophthalmology
Under oxidative stress, the apoptosis of TMCs was significantly elevated compared to the control condition; however, miR-486-5p overexpression led to a marked inhibition in cell apoptosis (Figure 2(a)). Western blotting also showed that miR-486-5p overexpression reduced the promotive effects of H 2 O 2 on the expression levels of cleaved caspase-3/ 9 in TMCs (Figure 2(b)). SOD is the main antioxidative metalloenzyme that is able to resist oxygen-free radicals; MDA is the final product of peroxidation [31]. We thus assessed the levels of SOD and MDA in TMCs under the treatment of H 2 O 2 . As presented in Figures 2(c) and 2(d), H 2 O 2 notably increased the SOD and MDA levels compared to the control, but these changes were reversed by miR-486-5p overexpression. We then evaluated the effects of miR-486-5p on ECM in H 2 O 2 -stimulated TMCs using the immunofluorescence staining assay and discovered that miR-486-5p overexpression could significantly downregulate ECM genes, including collagen I, fibronectin, and laminin expression (Figures 2(e)-2(g)). Western blotting further indicated the same effects of miR-486-5p overexpression on the expression levels of collagen I, fibronectin, and laminin (Figure 2(h)).

SMAD2 Downregulation Attenuated H 2 O 2 -Induced
Injury in TMCs. Next, we explored the effects of SMAD2 on H 2 O 2 -induced injury in TMCs. e sh-SMAD was transfected into TMCs to downregulate SMAD2. RT-qPCR showed that SMAD2 was knocked down by sh-SMAD2 compared to the control (sh-NC) (Figure 4(a)). Downregulated SMAD2 markedly decreased the apoptosis that was elevated by H 2 O 2 (Figure 4(b)). Western blotting showed reduced protein expression of cleaved caspase-3/9 in H 2 O 2 -treated TMCs after SMAD2 downregulation (Figure 4(c)). Moreover, SMAD2 downregulation abrogated the effects of H 2 O 2 on the SOD and MDA levels in TMCs (Figures 4(d) and 4(e)). ECM genes, including collagen I, fibronectin, and laminin expression, were significantly upregulated by H 2 O 2 , but this effect was abolished by SMAD2 downregulation (Figure 4(f )).

Discussion
e TGF-β/SMAD signaling pathway is required for fibrotic progress and ECM production, frequently detected in types of pathological conditions or diseases. For example, idiopathic pulmonary fibrosis has the characteristics of excessive fibroblast proliferation and excessive deposition of ECM components [32,33]. It was proven that protein tyrosine phosphatase α induces profibrotic signaling pathways via controlling cellular responses to TGF-β [34]. Emerging research has revealed that TGF-β is able to upregulate collagen-1 and fibronectin expression [35,36]. e SMAD system is the predominant intracellular signaling of TGF-β. In addition, other pathways, such as p38 MAPK and PI3K/ Akt, can respond to TGF-β and promote ECM synthesis via SMAD pathways [36,37].
TGF-β/SMAD signaling also plays a role in the TM, which is commonly overactive in glaucomatous neuropathy [9,38]. Both non-SMAD and SMAD pathways are involved in TGF-β-induced lysyl oxidase synthesis, which takes major responsibility for elevated intraocular pressure [39]. As the members of the SMAD system, SMAD2/3 are shown to be responsible for TGF-β2-induced ECM deposition in the TM [40]. Recent studies that have focused on the roles of miRNAs in modulating the biological functions of the TM contribute to identifying more biomarkers for the treatment of glaucoma [27,41]. It was demonstrated by Hubens et al. that hsa-miR-486-5p has a downregulated level in glaucoma patients [29]. In the lens, miR-486-5p targets SMAD2 to repress TGF-β2-induced ECM remodeling [30].     Figure 5: miR-486-5p regulated H 2 O 2 -induced injury in TMCs by targeting SMAD2. After H 2 O 2 -treated TMCs were transfected with NC mimics alone, miR-486-5p mimics alone, miR-486-5p mimics plus pcDNA3.1 alone, or miR-486-5p mimics plus pcDNA3.1/SMAD2, (a, b) mRNA and protein expression of SMAD2 was assessed by RT-qPCR and western blotting, respectively; (c) apoptosis was assessed by flow cytometry; (d) protein expression of cleaved caspase-3/9 was determined by western blotting; (e, f ) the SOD and MDA levels were assayed; and (g) expression of collagen I, fibronectin, and laminin was tested by western blotting. * p < 0.05; * * p < 0.01; * * * p < 0.001.
Additionally, miR-486-5p suppresses non-small cell lung cancer by targeting the TGF-β/SMAD2 signaling [42]. If miR-486-5p could target SMAD2 in TMCs, elevated ECM remodeling may be detected in glaucoma patients. is also may contribute to elevated production of fibronectin that is related to reduced aqueous humor outflow and increased stiffness [43]. erefore, investigating whether restoring miR-486-5p level can downregulate TGF-β/SMAD2 signaling in TMCs under oxidative stress, preventing ECM remodeling and cell death, is necessary. In this research, we demonstrated a targeted relationship between SMAD2 and miR-486-5p in TMCs under oxidative stress. miR-486-5p was downregulated and SMAD2 was upregulated by H 2 O 2 . Functionally, miR-486-5p upregulation inhibited apoptosis, reduced the MDA level, and increased the SOD level as well as downregulated ECM genes, showing the same function as SMAD2 knockdown. Moreover, SMAD2 upregulation abrogated the protective effects of miR-486-5p against H 2 O 2induced injury.
Overall, oxidative stress induces marked alternations in miRNA expression levels, which contributes to some cellular responsiveness to oxidative stress in TMCs. Concretely speaking, we demonstrated that miR-486-5p restrains oxidative stress-induced ECM synthesis via targeting SMAD in TMCs. erefore, the miR-486-5p/SMAD2 axis may act as a regulatory mechanism limiting ECM accumulation induced by oxidative stress in the aqueous humor outflow pathway. In future work, we would conduct animal experiments to persuade our findings.
Data Availability e datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.