Lens epithelial cells were collected as previously described (20) from 12 patients with cataracts and 12 normal controls receiving eye surgery at Beijing Tongren Hospital (Beijing, China). The study protocol followed the guidelines of the Declaration of Helsinki (21) and was approved by the institutional review board of Beijing Tongren Hospital. Following a full explanation of the surgical procedures and possible complications, all patients provided written informed consent. Patients were selected based on clinically observable nuclear cataracts of grade 2 or 3 according to the Lens Opacities Classification System III (22). Exclusion criteria included cataract hardness greater than grade 3. Patients with type 1 diabetes mellitus (DM), rheumatologic disease and other systemic diseases, with the exception of type 2 DM, were also excluded. SRA01/04 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 100 mg/ml streptomycin, 100 U/ml penicillin and 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂ at 37°C.

A DNA extraction kit (Qiagen, Inc., Valencia, CA, USA) was used to extract genomic DNA from patient lens cells. The Sanger method was used to bidirectionally sequence the TP53 3′-UTRs (Beijing Tongren Hospital). A TP53 reference sequence (NM_000546.5; genome.ucsc.edu) was compared with all sequences and used to determine the genotype of the polymorphism.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from cataractous lens samples and cultured SRA01/04 epithelial cells according to the manufacturer's instructions. A UV-Vis spectrophotometer (UV-1800; Shimadzu Corporation, Kyoto, Japan) was used to determine the quality of RNA. Agarose gel (1.5%) electrophoresis with 260/280 values between 1.8 and 2.0 was used to confirm RNA integrity. PrimerScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) was used produce cDNA from 1 ng RNA for RT-qPCR analysis. An ABI 7500 cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.), RT primer and TaqMan probes (Guangzhou RiboBio Co., Ltd., Guangzhou, China) were used to determine the expression of miR-125b and TP53. RNA U6 small nuclear 2 was used as the endogenous reference control. The conditions for the RT reaction were 65°C for 5 min, 25°C for 10 min, 42°C for 1 h and 75°C for 10 min. The PCR cycling conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 10 sec, and 60°C for 1 min. RT-qPCR analysis was performed twice on three independent specimens. The primer (Shanghai Jieli Biotechnology Co., Ltd., Shanghai, China) sequences were as follows: Forward, 5′-TCAGTTTGCTGTTCTGGGTG-3′ and reverse, 5′-CGGTTGGCTGGAAAGGAG-3′ for GAPDH; forward, 5′-CCCCTCTGAGTCAGGAAACA-3′ and reverse, 5′-AGACAGAAGGGCCTGACTCA-3′ for TP53; forward, 5′-TCAGTTTGCTGTTCTGGGTG-3′ and reverse, 5′-CGGTTGGCTGGAAAGGAG-3′ for U6; and forward, 5′-UCCCUGAGACCCUAACUUGUGA-3′ and reverse, 5′-ACAAGUUAGGGUCUCAGGCACU-3′ for miR-125b. The equation RQ=2^−ΔΔCq was used to calculate the relative abundance of miR-125b and TP53 mRNA in cell lines and tissues (23). GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to produce graphs and perform analyses.

Human genomic DNA was used for amplification of the 3′-UTR of the TP53 gene containing conserved binding sites for miR-125b. Following amplification, the fragments were implanted into the pmiR-RB-REPORT vector (Guangzhou RiboBio Co., Ltd.). A mutant 3′-UTR fragment containing mutations in seed binding sites was obtained using a QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's protocols, to introduce deletions in the 3′-UTR of TP53. Similarly, the TP53-3′-UTR mutant fragment was inserted into the same sites of the pmiR-RB-REPORT control vector. For reporter assays, SRA01/04 cells were plated in 24-well plates and cultured overnight in Iscove's modified Dulbecco's medium (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation, the cells were cotransfected with miR-125b mimic/mimic control and wild-type/mutant reporter plasmid (Guangzhou RiboBio Co., Ltd.) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, the Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA) was used to determine luciferase activity according to the manufacturer's protocol. The GloMax™ 96 Microplate Luminometer (Promega Corporation) was used to measure luciferase activity. The endogenous control was Renilla luciferase plasmid. Each experiment was conducted with three independent specimens and repeated twice.

At 48 h following transfection, an apoptosis assay was performed on SRA01/04 lens epithelial cells (1.5×10⁵ cells/well). An apoptosis detection kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China) was used to determine apoptosis using Annexin V/propidium iodide staining. The specimens were cultured at room temperature for 15 min in darkness and analyzed by flow cytometry (BD Influx™ Cell Sorter; BD Biosciences, Franklin Lakes, NJ, USA). CellQuest software (BD Biosciences) was used to analyze the results.

Protein extraction from SRA01/04 and primary cells was performed by incubation with lysis buffer containing 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 2 mg/ml aprotinin, 1 mM phenylmethane sulfonyl fluoride and 150 mM NaCl at 4°C for 30 min. A BCA Protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantify the protein levels. The separation of protein extracts (20 μg) was performed via 10% SDS-polyacrylamide gel electrophoresis (Roche Applied Science, Penzberg, Germany) and transferred onto polyvinylidene difluoride membranes (PerkinElmer, Inc., Waltham, MA, USA). Membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST; BioSharp, Hefei, China) with 5% non-fat milk, 2.7 mmol/l KCl, 137 mmol/l NaCl and 25 mmol/l Tris-HCl (pH 7.5) at 37°C for 1 h. The membranes were then incubated with primary antibodies, mouse monoclonal against β-actin (1:8,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-47778) or rabbit polyclonal against TP53 (1:3,000; Santa Cruz Biotechnology, Inc.; cat. no. 6243) in TBST with 5% non-fat milk at 4°C overnight. Following washes with TBST three times, membranes were cultured with horseradish peroxidase-conjugated goat anti-rabbit polyclonal (1:15,000; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. Q11402MP) and goat anti-mouse (1:10,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-2005) secondary antibodies for 1 h at room temperature. Membranes were washed again with TBST, then observed using a SuperSignal West Pico Chemilumunescent Substrate enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and analyzed with ImageJ 1.48 (imagej.nih.gov/ij/).

The differences in microarray data were compared between the two groups using the P-values corrected by false discovery rate, which were below 0.05 obtained by significant analysis of microarrays software (statweb.stanford.edu/~tibs/SAM/). SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA) and an independent samples t-test were used to estimate differences between two groups. P<0.05 was considered to indicate a statistically significant difference.

miRNAs negatively regulate the expression of target genes by binding to the ʻseed sequence' in the gene 3′-UTR. Variants within or nearby the ʻseed sequences' may compromise or enhance miRNA/mRNA interaction leading to either ʻloss-of-function' or ʻgain-of-function' effects. The present study evaluated the potential effect of the rs78378222 polymorphism in the 3′-UTR of TP53 on miRNA-125b-induced apoptosis of lens epithelial cells, and the association between the rs78378222 polymorphism and the risk of age-associated cataracts. Initially, sequences of mature miR-125b and TP53 (wild-type and polymorphic) were analyzed and compared. As presented in Fig. 1, replacement of A with C (at position 1177) introduces a novel potential binding site in the 3′-UTR of TP53 with consecutive 8-bp perfect match, indicating that the rs78378222 polymorphism results in a ʻgain-of-function' to regulate TP53 expression.

To examine the effect of the rs78378222 polymorphism on TP53 expression, the full length TP53 3′-UTR was subcloned and inserted into a pcDNA3 vector containing a firefly luciferase gene downstream, and the minor allele of the rs78378222 polymorphism (C) was introduced. The results of the luciferase assay demonstrated that transfection with wild type TP53 3′-UTR (A) significantly reduced the luciferase activity of the miR-125b overexpressing cells, compared with the scramble controls. Furthermore, the miR-125b overexpressing cells transfected with the construct containing the minor allele of rs78378222 polymorphism (C) was reduced further than those transfected with wild type 3′-UTR, suggesting miR-125 negatively regulates the expression of TP53 by targeting the 3′-UTR of the gene (P<0.01; Fig. 2), and introduction of a novel binding site caused by the rs78378222 polymorphism further promoted the negative regulatory association between miR-125 and TP53.

To further confirm this hypothesis, epithelial cells were collected from patients with age-associated cataracts and controls, and the mRNA expression levels of miR-125b and TP53 were determined. As demonstrated in Fig. 3, the expression level of miR-125b was comparable between the two groups, whereas the mRNA expression level of TP53 was significantly higher in the cataract group compared with the control (P<0.01). Additionally, the expression of TP53 protein was determined using western blot analysis (Fig. 4). The target bands were quantified by analyzing their relative density, which demonstrated that the protein expression level of TP53 was significantly higher in the cataract cases compared with the control group (P<0.01; Fig. 4).

To further investigate the regulatory association between miR-125b and TP53, miR-125b mimics were transfected into cultured SRA01/04 epithelial cells. Transfection with 25 nM mimics was not identified to significantly alter the expression of TP53; however, 50 nM miR-125b mimics substantially reduced the mRNA and protein expression levels of TP53 in the lens epithelial cells (P<0.01; Fig. 5). Additionally, compared with negative controls, miR-125b (50 nM) significantly induced apoptosis in the epithelial cells (Fig. 6).