The present study was approved by the Ethical Committee of the Fourth Affiliated Hospital of China Medical University. Written informed consent was obtained from each patient. Age-related cataract patients without other ocular diseases undergoing cataract surgery (phacoemulsifcation) were enrolled at the Fourth Affiliated Hospital of China Medical University (Shenyang, China). Normal eyes were obtained from the Eye Bank of the Fourth Affiliated Hospital of China Medical University. Fresh anterior lens capsules isolated from age-related cataract patients and normal eyes were immediately frozen in liquid nitrogen at the time of surgery and stored at −80°C.

Human lens epithelial cell line cells (SRA01/04 cells, a kind gift of Dr. Yi-sin Liu, Doheny Eye Institute, Los Angeles, CA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) in a humidifed incubator at 37°C with 5% CO₂.

Total RNA was extracted from tissues and cells using the Trizol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For RT-qPCR analysis of miR-24, the total RNA isolated from cells was subsequently reverse transcribed to cDNA using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression of miR-24 was determined using TaqMan MicroRNA assays (Applied Biosystems; Thermo Fisher Scientific, Inc.), and standardized to RNU6B expression. The upstream and downstream primers of miR-24 and RNU6B were purchased from Thermo Fisher Scientific, Inc., and their sequences can be found on their website. For RT-qPCR analysis of p53, cDNAs were obtained using the PrimerScript RT reagent kit (Takara Bio Inc., Tokyo, Japan), RT-qPCR analysis of p53 was performed with TaqMan Universal Master Mix II (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primers for p53 were as follows: Forward 5′-CAGCAGTCAAGCACTGCCAAG-3′, reverse 5′-AGACAGGCATGGCACGGATAA-3′, and β-actin was used for normalization, β-actin primer sequences were: Forward: 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, Reverse: 5′-ATGGAGCCACCGATCCACA-3′. The RT-qPCR analysis was performed on ABI 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.). All experiments were performed in triplicate. The relative expression levels of mRNA or microRNA were calculated using the 2^−ΔΔCt method.

Total protein was extracted using a RIPA lysis buffer supplemented with a protease inhibitor cocktail (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts (40 µg) of proteins were separated using NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen; Thermo Fisher Scientific, Inc.), then transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% fat-free milk at room temperature for 2 h and incubated with the primary antibodies including rabbit anti-p53 (1:1,000; Abcam, Cambridge, CA, USA) and rabbit anti-GAPDH (1:2,000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:2,500; Promega Corporation, Madison, WI, USA) at room temperature for 2 h. The protein bands were visualized using the an ECL Western Blotting Substrate kit (Pierce; Thermo Fisher Scientific, Inc.) and quantified using Image J software (National Institute of Health, USA).

A 2′,7′-dichloro-fluorescein diacetate (DCFH-DA) probe was used to detect fluorescence derived from endogenous ROS in hLECs. 1×10⁴ cells were seeded into each well of a 96-well plate and were cultured for 16 h, until cells were observed adhering to the sides of the well. The cells were then exposed to different concentrations H₂O₂ (0, 100, 200, 400, 600, 800, 1,000 µM) for 1 h whereupon the culture medium was aspirated and 10 uM fluorescent probe DCFH-DA was added to each well. This mixture was then incubated in a 37°C incubator for 20 min. The cells were then washed three times with PBS and their DCF fluorescence intensity value (i.e., the mean fluorescence intensity of DCF, representing the level of intracellular ROS) was read using a multifunctional microplate reader. The excitation wavelength used was 485 nm and the emission wavelength was set at 530 nm.

Cell viability and proliferation were determined using the CellTiter96AQ_ueous One Solution Cell Proliferation assay kit (Promega). The reagent contains a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS). After treatments, according to the manufacturer's protocols, 20 µl MTS solution was added to each well of the 96-well assay plate containing the cells in 100 µl of culture medium and the cells were then incubated for 4 h at 37°C, 5% CO₂. The absorbance of each group was read using an absorbance plate reader set to a 490 nm wavelength. The cell viability rates were calculated according to the following formula: The cell viability ratio (%)=[(As-Ab)/(Ac-Ab)]x100%, where As is the optical density value at 490 nm (OD490) of H₂O₂ treatment group, Ab is the OD490 of blank group, and Ac is the OD490 of non-H₂O₂ control group. Each experiment was repeated three times.

Caspase-3 activity was detected using a caspase-3 assay kit (Abcam). After treatments, in accordance with the manufacturer instructions, these SRA01/04 cells were lysed in 50 µl of chilled Cell Lysis buffer and incubated on ice for 10 min, centrifuged, the supernatant protein concentration was determined using the BCA method. 50 µl of Cell Lysis buffer containing 100 µg protein were added to each well in a 96 well plate. Then, 50 µl 2× Reaction buffer, 0.5 µl 10 mM DTT and 5 µl caspase-3 catalytic substrate DEVD-p-NA substrate were added to each well. The samples were incubated at 37°C for 2 h. The optical density (OD) value was obtained using a microplate reader set at 405 nm wavelength. Each experiment was repeated three times. In the caspase-3 activity control group, OD was set to a value of 1 and the caspase-3 experimental group activity was standardized using the following calculation: (OD value of experimental group-blank well OD)/(OD value of control group-blank well OD) ×100%.

The miR-24 mimic (miR-24), mimic negative control (miR-Ctrl), miR-24 inhibitor (anti-miR-24), inhibitor negative control (anti-miR-Ctrl), small interfering RNA for p53 (p53 siRNA) and siRNA control were purchased from GenePharma, Inc., (Sunnyvale, CA, USA). SRA01/04 cells were seeded in a 6-well plate, and transfection was conducted after 24 h. Transfections were performed according to the manufacturer's instructions with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 72 h, the cells were treated with 400 µM H₂O₂ for 1 h after which the expression of miR-24 was measured using RT-qPCR. The expression of p53 was measured using RT-qPCR and western blotting, and the cell viability was measured using MTS.

We used human cDNA to generate wild-type and mutant 3′-UTR sequences for the p53 gene, including predicted miR-24 targeting regions. We then cloned these amplified fragments into the pGL3-Promoter vector (Promega) at the same location. For luciferase reporter assays, these reporters were cotransfected into SRA01/04 cells together with miR-24 mimics and mimic controls. Luciferase activity was then evaluated using a Dual-Luciferase Reporter Assay System kit (Promega) at 72 h after transfection. The Renilla luciferase plasmid was used as an endogenous control. The experiments were performed in triplicate.

Each experiment was repeated independently at least 3 times with similar results. Measurement data were presented as mean ± standard deviation (SD). Differences between two groups were calculated using an independent sample t-test. Differences among multiple groups were determined by one-way analysis of variance followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was done using SPSS v16.0 (SPSS, Inc., Chicago, IL, USA).

One previously conducted microarray study observed that miR-24 levels dramatically changed in human cataractous lenses (18), but this finding has not been verified. To explore the expression levels of miR-24 in the lens epithelial cells (LECs) of age-related cataracts, RNAs isolated from the 48 anterior lens capsules of age-related cataract patients and the 32 normal anterior lens capsule specimens were subjected to RT-qPCR analysis. The assays showed that the expression of miR-24 was significantly increased in cataract tissues as compared to normal tissues (Fig. 1). These results suggest that miR-24 may play a role in cataract development.

p53 has been implicated as an important protein in cell proliferation and differentiation during embryonic lens development (19). In the present study, the expression of p53 was also examined by RT-qPCR and western blotting in anterior lens capsules (control: n=44, cataract: n=56). Both the expression of p53 protein and p53 mRNA were found to be significantly up-regulated in the cataract tissues when compared with the normal tissues (Fig. 2). This finding, combined with the previous miR-24 experiment (Fig. 1) demonstrates a positive correlation between endogenous miR-24 and p53 expression.

In order to establish the expression levels of miR-24 and p53 in LECs exposed to oxidative stress, we treated SRA01/04 cells with 400 µmol H₂O₂ for 1 h. The expression of miR-24 was detected using RT-qPCR which showed a great increase in SRA01/04 cells exposed to H₂O₂ compared with controls (Fig. 3A). RT-qPCR (Fig. 3B) and western blotting analysis of p53 (Fig. 3C and D) revealed a significant increase of p53 in SRA01/04 cells treated with H₂O₂. This increase was correlated with higher expression of ROS (Fig. 3E), decreased cell viability (Fig. 3F) and increased caspase-3 activity (Fig. 3G). This suggests that ROS modulated both miR-24 and p53 in LECs.

To investigate the correlation between miR-24 and p53 expression in LECs exposed to oxidative stress, we transfected SRA01/04 cells with either miR-24 mimics or inhibitors, then the medium was removed and 400 µmol H₂O₂ was added to induce oxidative stress. RT-qPCR was performed to assess the level of p53-mRNA expression. As is shown in Fig. 4A and B, samples with overexpressed miR-24 up-regulated p53 mRNA expression when compared with controls, while inhibition of miR-24 down-regulated the expression of p53 mRNA. Western blotting indicated that p53 protein levels were enhanced in SRA01/04 cells transfected with miR-24 mimics and reduced in cells exposed to miR-24 inhibitors (Fig. 4C-F). These results indicated that the expression of p53 was regulated at both the mRNA and protein level by miR-24 in LECs.

To more closely examine the mechanisms of miR-24 and p53 in cataracts, we used bioinformatics with publicly available databases (TargetScan, miRanda and miRBase) to determine whether p53 may be the target of miR-24 (Fig. 5A). To confirm the targeting of p53 by miR-24, luciferase activity assays were performed. SRA01/04 cells were co-transfected the luciferase reporter construct pGL3-p53-wt or pGL3-p53-mut with miR-24 mimics. As shown in Fig. 5B, SRA01/04 cells with pGL3-p53-wt and miR-24mimics had significantly increased reporter activity when compared with the controls, whereas no significant difference in reporter activity was observed when the target site was mutated. Together, these results indicate that 3′UTR of p53 carries a direct and functional binding site for miR-24 in LECs.

To identify the role of miR-24 in LEC viability and apoptosis, we used MTS assays to measure the viability of LECs, and caspase-3 activity was also assessed. The SRA01/04 cells were transfected with miR-24 mimics, mimic controls, miR-24 inhibitors and inhibitor controls before exposure to H₂O₂ (400 µmol). As assayed by MTS, SRA01/04 cells transfected with miR-24 mimics displayed significantly increased cell death compared to those transfected with mimic controls, while transfection with miR-24 inhibitors significantly suppressed H₂O₂-induced LEC death (Fig. 6A). Results of caspase-3 activity shown that compared with the control group, the miR-24 mimic group had significantly elevated caspase-3 activity while the caspase-3 activity of the miR-24 inhibitor group was markedly decreased (Fig. 6B). These results suggest that miR-24 promotes apoptosis and inhibits the proliferation of human LECs exposed to oxidative stress.

The previous experiments clearly indicated that miR-24 enhances LEC death. To explore whether p53 is involved in miR-24 enhanced LEC death and apoptosis, we knocked down p53 using p53 siRNA. Before exposure to H₂O₂, SRA01/04 cells were transfected with p53 siRNA in the absence or presence of miR-24 mimics. 72 h after transfection, cell viability was assessed by MTS assays, caspase-3 activity was assessed by caspase-3 activity assays and the protein expression of p53 was measured by western blotting. Co-transfection of miR-24 mimics with p53 siRNA significantly reversed the p53 protein expression induced by miR-24 mimics transfection (Fig. 7A and B). As shown in Fig. 7C and D, when compared with cells co-transfected with the mimic controls and the siRNA controls, the viability and caspase-3 activity of LECs co-transfected with miR-24 mimics and p53 siRNA showed little change. In addition, knockdown of p53 in LECs resulted in an increased cell survival rate and decreased caspase-3 activity. These results indicate that miR-24 enhanced LEC death and apoptosis by directly targeting p53.