The HTM and GTM cells were provided by Zhongshan Ophthalmic Center of Sun Yat-sen University (Guangzhou, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 20% fetal bovine serum in a humidified atmosphere with 5% CO2 at 37°C. The cells were passaged when their confluence reached 70% and were washed twice with D-Hanks solution.

The lentiviral vectors specific for pre-miR-93 and miR-93 sponge were constructed by GenePharma (Shanghai, China). Prior to transfection, the GTM cells were transferred to 24-well plates (1×10⁵ cells/well) and cultured for 24 h. The lentivirus suspension was added to the cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol, and blank lentivirus was used in the control group. Following incubation for 24 h at 37°C, the suspension in the wells was replaced with DMEM/F12 medium and the cells were cultured for another 48 h at 37°C. The transfection efficiency was monitored using a fluorescence-activated cell sorter (BD FACSCanto II; BD Biosciences, San Jose, CA, USA), and the cells were collected for further analyses.

The transfected GTM cells were adjusted to the concentration of 5×10⁴/ml, transferred to 96-well plates (200 µl per well), and cultured at 37°C for 24, 48, 72 and 96 h. Fresh medium containing 0.5 mg/ml methyl thiazolyl tetrazolium (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was added to each well and the cells were incubated at 37°C for 4 h, following which the reaction was terminated using 200 µl dimethyl sulfoxide. The optical density at 570 nm was detected for each well using a microplate reader (Molecular Devices LLC, Silicon Valley, CA, USA).

The transfected GTM cells were transferred to 24-well plates 48 h following transfection, at a density of 2×10⁵ cells per well. Cell apoptosis was analyzed using the annexin V-fluorescein isothiocyanate (FITC)/prodium iodide (PI) dual staining method with an Annexin V:FITC Apoptosis Detection kit II (BD Biosciences) according to the manufacturer's protocol. The cells were then analyzed using the BD FACSCanto II. The percentages of apoptotic cells (annexin V positive and PI negative) were compared.

The microRNAs of the HTM and GTM cells were extracted using RNAiso for small RNAs (Takara Biotechnology Co., Ltd., Dalina, China) and reverse transcribed using a One Step PrimeScript miRNA cDNA synthesis kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed with SYBR Green I Mastermix (Roche Diagnostics GmbH, Mannheim, Germany) using a LightCycler 480 (Roche Diagnostics, Basel, Switzerland). The total reaction volume was 10 µl which consisted of 0.25 µM each primer and approximately 5 ng of template cDNA. Accordingly, the thermocycling profiles were as follows: Denaturation at 95°C for 5 min; 45 cycles of 95°C, 60°C for 20 sec and 72°C for 20 sec, the melt curve protocol was 95°C for 5 sec, 65°C for 1 min and reheating to 97°C. Finally, the temperature was reduced to 40°C and maintained for 10 sec. The specific primers for miR-93 was synthesized as forward 5′-AGTCTCTGGCTGACTACATCACAG-3′ and reverse 5′-CTACTCACAAAACAGGAGTGGAATC-3′. U6 (forward 5′-CTCGCTTCGGCAGCACA-3′ and reverse 5′-AACGCTTCACGAATTTGCGT-3′) was used as the internal control. Data were analyzed using the 2^−ΔΔCq method (16).

The online tool microRNA.org (www.microrna.org/microrna/home.do) was used to predict the target gene for miR-93, and was used to select the specific primary antibody for the present study. Protein samples of the infected GTM cells were prepared using RIPA lysis buffer (Beyotime Institute of Biotechnology, Inc., Shanghai, China). The protein contents were quantified using the BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). A protein sample of 20 µg was loaded in each lane for separation of the samples by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following which they were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and incubated with anti-NFE2L2 (monoclonal; rabbit anti-human; 1:1,000; cat. no. ab62352) or anti-GAPDH (internal reference; monoclonal; rabbit anti-human; 1:10,000; cat. no. ab181602) primary antibodies (Abcam, Cambridge, UK) at 4°C overnight. Following washing with a buffer containing 0.1% Tween-20, 20 mM Tris-HC and 137 mM NaCl (TBST), the membrane was incubated in horseradish peroxidase-conjugated secondary antibody (polyclonal; goat anti-rabbit; 1:10,000; cat. no. ab97051) for 1 h at room temperature. Signals were developed using ECL Plus western blotting substrate (Thermo Fisher Scientific, Inc.) and the intensity of bands was analyzed using the ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

All experiments were repeated in triplicate, and results are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 19.0 software (IBM SPSS, Armonk, NY, USA) using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Prior to investigating the functions of miR-93, the expression levels of miR-93 in HTM and GTM cells were compared using RT-qPCR analysis (Fig. 1). The expression of miR-93 was significantly upregulated in the GTM cells, which was almost four times higher, compared with that in the HTM cells (P<0.01). This indicated the association between miR-93 and glaucoma; therefore, it was necessary to reveal the roles of miR-93 in GTM.

To investigate the role of miR-93 in GTM cells, miR-93 was inhibited and overexpressed by transfection with its specific inhibitor sponge and expression vector, respectively. Alterations in the expression of miR-93 were analyzed using RT-qPCR analysis (Fig. 2). The results indicated the successful inhibition and overexpression of miR-93, with its expression levels significantly reduced following miR-93 sponge transfection (P<0.001), and recovered to the original level following co-transfection with the miR-93 sponge and pre-miR-93 vector (P<0.01). Therefore, these three groups of transfected cells were suitable for use in the following experiments.

GTM cell viability was monitored during the 96 h time period following transfection (Fig. 3A). The viability of the GTM cells in the control group gradually decreased from 48 h post-transfection. By contrast, the cells with inhibited miR-93 showed marked promotion of cell viability, which was significant higher, compared with that in the control group (P<0.01). The results also showed that cell viability was inhibited when miR-93 was overexpressed (P<0.05), which indicated that miR-93 was capable of inhibiting GTM cell viability. At 96 h post-transfection, the percentages of apoptotic cells were also examined using annexin V-FITC/PI staining (Fig. 3B). Compared with the control group, cell apoptosis was inhibited when miR-93 was suppressed (P<0.001). However, in the cells overexpressing miR-93, the effects of the miR-93 sponge was abrogated, with the percentage of apoptotic cells significantly increased (P<0.001), and an increase in cells in the late stage of apoptosis. Taken together, these results indicated that miR-93 induced the apoptosis of GTM cells and inhibited their viability, suggesting it is an important regulator in glaucoma.

The present study subsequently investigated the regulatory mechanism of miR-93. As a previous study reported that NFE2L2 is a target gene of miR-93 during breast carcinogenesis (17), the protein expression levels of NFE2L2 were analyzed in the transfected GTM cell groups in the present study (Fig. 4). The results indicated that the expression of NFE2L2 was promoted in the GTM cells with inhibited miR-93 (P<0.01) and was suppressed when miR-93 was overexpressed in the cells (P<0.01). These results suggested that miR-93 inhibited NFE2L2 in the GTM cells, which may be the possible functional pathway underlying the effect of miR-93 on regulating glaucoma.