MiR-181 a inhibits human trabecular meshwork cell apoptosis induced by H2O2 through the suppression of NF-κB and JNK pathways

Results. The apoptosis assay and qRT-PCR showed that H2O2-induced apoptosis and cell viability were suppressed in a dose-dependent manner in TM cells. After the TM cells were treated with H2O2, miR-181a expression was significantly lower. The overexpression of miR-181a enhanced TM cells’ viability, while the knockdown of miR-181a inhibited viability of cells. The overexpression of miR-181a suppressed TM cell apoptosis, while the knockdown of miR-181a induced apoptosis. H2O2 activated the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways and induced cell apoptosis, while the overexpression of miR-181a suppressed both pathways and decreased the rate of apoptosis.


Introduction
Worldwide, glaucoma is the one of the leading causes of irreversible blindness. 1,2There are 60 million people diagnosed with glaucoma, of whom 8.4 million have been blinded due to glaucoma. 2One of the major risk factors associated with glaucoma includes elevated hydrostatic pressure or intraocular pressure (IOP) in the eyes. 3Intraocular pressure depends on the rate of formation and drainage of the aqueous humor via the trabecular meshwork. 3he trabecular meshwork (TM), consisting of TM cells and located at the intraocular anterior chamber angle, plays a critical role in the outflow of aqueous humor. 4Decreased outflow of aqueous humor induces an elevation in IOP and ultimately leads to glaucoma. 3,4This in turn leads to increased oxidative DNA damage in the TM of glaucoma patients.The oxidative stress induces the activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling pathways. 5Trabecular meshwork cells play an important role in maintaining the extracellular matrix synthesis (ECM) and phagocytosis of the debris in outflow tissues which control the aqueous-outflow facility.Thus, a shortage of TM cells might result in ocular hypertension.Therefore, the elucidation of the mechanism underlying the miR-181a and primary oxidative stress response signaling pathways might reveal promising opportunities for preventing TM cell apoptosis, which can prevent glaucoma. 5An understanding the biological functions of TM cells is then beneficial for understanding the pathogenesis of glaucoma and for developing novel drugs.
MicroRNAs (miRNAs) are a group of non-coding RNAs with 14-20 nucleotides, which are involved in the inhibition of endogenous gene expression through translational cleavage. 6][9][10] These miRNAs, either directly or indirectly, have been found to play an important role in the pathogenesis of glaucoma in both animal and human visual systems. 11,12The role of miRNAs in the molecular mechanisms of ECM provides potential for the development of drugs. 13In their study, Dismuke et al. isolated 10 mature miRNAs from human aqueous humor exosomes, one of which was miR-181a. 14miR-181a is a family member of miR-181, and has been reported to act as a regulator in cell proliferation, migration and invasion. 15Interestingly, miR-181a has different functions in different types of cells.7][18] The levels of miR-181a were most decreased with aging. 19The exact underlying mechanism of miRNAs regulate retinal apoptosis or degeneration is largely unknown.In a study by Parikh et al., miR-181a was found to promote transdifferentiation towards mesenchymal-like cells. 20The activation of these pathways is thought to contribute to the dysfunction of normal outflow facility. 21so, a study by Kozloski et al. demonstrated that miR-181a negatively regulated NF-κB activity, leading to a significant change in the proliferation and survival phenotype of the B-cell-like diffuse large B-cell lymphoma malignancy. 22owever, the role of miR-181a in the pathogenesis of glaucoma remains elusive.
In this study, we investigated the role of miR-181a in apoptosis in human trabecular meshwork cells induced by H 2 O 2 through the NF-κB and JNK pathways.

Cell culture and H 2 O 2 treatment
Human primary explant-derived trabecular meshwork (TM) cells (TM cell line) were obtained from ScienCell Research Labs (Catalog No. 6590; Carlsbad, USA).Primary human TM cells were grown in the Fibroblast Medium (Catalog No. 2301; ScienCell Research Labs, Carlsbad, USA) wherein the cells from the 3 rd to 6 th passage were used.For maintenance, the cells were incubated at 37°C in a 5% CO 2 environment.The TM cells were seeded in a 6-well plate or a 96-well plate and treated with different concentrations of H 2 O 2 for 2 h.

Small interfering RNA transfection
Mature miR-181a mimic and si-miR-181a were designed and synthesized by GenePharma (Shanghai, China).The insertion fragment was confirmed by DNA sequencing.Cell transfection and co-transfection were performed using the Lipofectamine 3000 reagent (Invitrogen Life Technologies, Carlsbad, USA) according to the manufacturer's instructions.

Apoptosis assay
Flow cytometry analysis was performed to identify and quantify the apoptotic cells by using an Annexin V-FITC/ PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China).The cells (100,000 cells/well) were seeded in a 6-well plate.The treated cells were washed twice with cold PBS and resuspended in buffer solution.The adherent and floating cells were combined and treated according to the manufacturer's instructions and measured with a flow cytometer (Beckman Coulter, Fullerton, USA) to differentiate apoptotic cells (Annexin-V-positive and PI-negative) from necrotic cells (Annexin-V-positive and PI-positive).

Quantitative reverse transcriptase--polymerase chain reaction
Total RNA was isolated from transfected cells by using TRIzol reagent (Invitrogen, Carlsbad, USA) and treated with DNase I (Promega, Southampton, UK).Reverse transcription was performed by using a Multiscribe RT kit (Applied Biosystems, Foster City, USA) and random hexamers or oligo(dT).The reverse transcription conditions were 10 min at 25°C, 30 min at 48°C, and a final step of 5 min at 95°C.

Western blot
The protein used for western blotting was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Guangzhou, China).The proteins were quantified using a BCA ™ Protein Assay Kit (Pierce, Appleton, USA).The western blotting system was established using a Bio-Rad Bis-Tris Gel system according to the manufacturer's instructions.Primary antibodies were prepared in 5% blocking buffer at a dilution of 1:1,000.Primary antibodies were incubated with the membrane at 4°C overnight, followed by washing and incubation with secondary antibodies marked by horseradish peroxidase for 1 h at room temperature.After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying blots and antibodies were transferred into the Bio-Rad ChemiDoc ™ XRS system, and then 200 μL Immobilon Western Chemiluminescent HRP Substrate (Millipore, Bedford, USA) was added to cover the membrane surface.The signals were captured and the intensity of the bands was quantified using Image Lab ™ Software (Bio-Rad, Shanghai, China).

Statistical analysis
All data were shown as mean ±SD.Statistical analyses were performed using SPSS 19.0 software (SPSS Science, Chicago, USA).Differences between groups were analyzed using one-way analysis of variance (ANOVA) and/ or Student's t-test.A p-value of <0.05 was considered statistically significant.All experiments were conducted in triplicate.

Different concentrations of H 2 O 2 suppressed viability of TM cells in a dose-dependent manner
In our study, we cultured TM cells in a 6-well plate or a 96-well plate and treated them with different concentrations of H 2 O 2 (50, 100, 200, and 300 μM) for 2 h.Cell viability was assessed by MTT assay.Our results showed that H 2 O 2 at a concentration of 200 μM was significantly suppressed.These results suggested that different concentrations of H 2 O 2 suppressed the viability of TM cells in a dose-dependent manner and that H 2 O 2 in increasing concentrations (100, 200, and 300 μM) suppressed cell viability significantly (Fig. 1, p < 0.05 or p < 0.01).For the analyses below 200 μM was selected.

H 2 O 2 -treated TM cells revealed a significant fall in miR-181a expression level
The TM cells after treatment with H 2 O 2 showed a significant decrease (p < 0.01) in the expression level of miR-181a (Fig. 2).

Transfection efficiency of TM cells
TM cells were transfected and then divided into mature miR-181a mimic, si-miR-181a and negative control (siNC) which were then evaluated by qRT-PCR.The results showed a significant decrease (p < 0.01) in the mRNA expression level of si-miR-181a compared to the control.Furthermore, they showed a significantly increased (p < 0.01) mRNA expression level of miR-181a mimic compared to the control.This suggested that transfection efficiency was observed when miR-181a was overexpressed or suppressed in TM cells (Fig. 3).

H 2 O 2 treatment and overexpression of miR-181a increased TM cell viability
Cell viability was measured in the TM cells after treatment with H 2 O 2 , and the effect of miR-181a on cell viability was measured after transfection.The results showed that cell viability was promoted with increasing time in the miR-181a mimic group compared to si-miR-181a and to control groups.These results suggest that the overexpression of miR-181a enhanced TM cell viability, while the knockdown of miR-181a inhibited cell viability (Fig. 4).

H 2 O 2 treatment and overexpression of miR-181a decreased TM cell apoptosis
Cell apoptosis was measured in the TM cells after treatment with H 2 O 2 , and the effect of miR-181a on cell apoptosis was measured after transfection.The results showed an increased percentage of cell apoptosis (p < 0.01) in the si-miR-181a group (mean: 35.7) compared to control groups.Treatment with H 2 O 2 caused nuclear condensation, which is an indicator of apoptosis.Also, the miR-181a mimic group (mean: 10.7) showed a significant reduction on cell apoptosis rate (p < 0.05), but not as significant as si-miR-181a.These results suggested that the overexpression of miR-181a suppressed TM cell apoptosis, while the knockdown of miR-181a induced apoptosis (Fig. 5).

miR-181a suppressed TM cell apoptosis via blocking the NF-κB and JNK pathways
Generally, H 2 O 2 activates the NF-κB and JNK pathways and induces cell apoptosis.The results showed that the si-miR-181a group has a decreased expression of miR-181a and treatment with H 2 O 2 suppresses the expression of miR-181a (p < 0.05).Thus, in the si-miR-181a + H 2 O 2 group, the expression of miR-181a is low and it cannot block the NF-κB and JNK pathways -it will rather produce just the opposite effect (Fig. 6).This suggested that the overexpression of miR-181a could suppress both the pathways, thereby decreasing the rate of cell apoptosis.

Discussion
Oxidative stress has been established as an important contributing factor in TM cell degeneration. 5The contributory role of H 2 O 2 in TM cell degeneration is also known. 23n the present study, we induced TM cell apoptosis through H 2 O 2 and found that miR-181a was downregulated by H 2 O 2 in a dose-dependent manner.It has been commonly shown in the literature that an aberrant expression of miR-181a has been associated with many types of human cancers and acts as a key oncogenic regulator. 23The underlying mechanism of miR-181a protects the TM cells is not quite clear.In the present study, we not only demonstrated an active change in the expression profile of miR-181a upon H 2 O 2induced apoptosis, but also a protective role of the miR-181a overexpression on TM cell.Thus, these results highlight the functional role of miR-181a in glaucoma.
This study explored the mechanisms of the regulation of miR-181a in TM cell injury.We found that the expression of miR-181a was downregulated in TM cells pretreated with H 2 O 2 .MTT results showed that the overexpression of miR-181a could improve cell viability, while the knockdown of miR-181a suppressed cell viability.Flow cytometry results demonstrated that miR-181a inhibited cell apoptosis, while the knockdown of miR-181a increased apoptosis.Western blotting assay showed a suppression of miR-181a cell apoptosis via blocking the NF-κB and JNK signaling pathways.
In this study, we also showed that during H 2 O 2 -induced apoptosis downregulating miR-181a blocks the NF-κB and JNK signaling pathways.Our results were similar to one of the studies conducted by Kozloski et al. in B-cell-like diffuse large B-cell lymphoma. 22Thus, it is likely that miR-181a upregulation may serve a protective role in glaucoma by exerting a protective effect on H 2 O 2 -induced apoptosis in TM cells.This hypothesis was further supported by our results, which showed that miR-181a overexpression promoted TM cell apoptosis, which possibly induced the transdifferentiation of mesenchymal-like cells in the aqueous humor of TM cells. 20NF-кB is considered to be an important nuclear transcription factor that helps in mediating the inflammatory response caused by oxidative stress. 24n endothelial cells, NF-кB is strongly activated by treatment with H 2 O 2 for 4 h. 25However, to elucidate the exact molecular interaction between miR-181a and the NF-κB and JNK pathways in protecting trabecular meshwork cells, future experiments that require blocking of these pathways or its upstream pathways upon the inhibition of miR-18a would provide solid evidence about the direct targeting of miR-181a on anti-apoptotic pathways in TM cells.
Many miRNAs are involved in the molecular mechanisms of glaucoma; some miRNAs were up-regulated in cells, while others were down-regulated. 12These miRNAs facilitate the evaluation of specific pathophysiology and offer diagnosis.Further in-depth understanding will help in diagnosing the subtypes of glaucoma, the pathophysiological stages, and the prescription of appropriate drugs. 12miR-183 was involved in the alteration of integrin-β1 expression, thereby effecting the physiology of TM, resulting in glaucoma. 26he miR-29 family regulates ECM synthesis in the TM. 27he changes associated with ECM synthesis result in alterations in the IOP.miR-29b negatively regulates the ECM proteins, and its regulators -under chronic oxidative stress conditions -affect ECM homeostasis in human TM cells.The expression of miR-29b decreased under chronic oxidative stress conditions, resulting in ECM gene upregulation.Similarly, miR-24 plays an important role in the flow pathways. 28One study identified the involvement of microRNAs in glaucoma with hsa-let-7b-3p as the most prevalent factor in the aqueous humor. 13n conclusion, this study indicated that miR-181a could improve the survival rate of TM cells after H 2 O 2 treatment by blocking the NF-κB and JNK signaling pathways.These findings might provide novel therapeutic oportunities in the treatment of glaucoma.

Fig. 1 .
Fig. 1.TM cell viability was significantly suppressed by H 2 O 2 in a dosedependent manner, and 200 μM was selected as a lethal concentration of 50% used in the following analyses * p < 0.05; ** p < 0.01.

Fig. 4 .
Fig. 4. The overexpression of miR-181a enhanced TM cell viability, while the knockdown of miR-181a inhibited cell viability

Fig. 6 .
Fig. 6.H 2 O 2 activated NF-κB and JNK pathways and induced cell apoptosis, while the overexpression of miR-181a suppressed both pathways and decreased the apoptotic rate * p < 0.05.