2. 1. Clinical Specimens
The anterior capsule specimens were collected from 14 senile cataract patients at Tianjin Eye Hospital using complete continuous circular capsulorhexis. Patients without diabetes were assigned to the control group, while patients with type 2 diabetes were assigned to the DC group. Patients with a history of eye surgery or other eye diseases, such as diabetic retinopathy, were excluded from this study. Before collecting the specimens, consent was obtained from all participating patients. The study was approved by the Research and Ethics Committee of Tianjin Eye Hospital and conducted in accordance with the principles of the Declaration of Helsinki.
2. 2. Cell Culture and Transfection of MicroRNA
HLEC-SRA01/04 cells (Saiku Biotechnology, Guangzhou, China) were incubated in normal glucose DMEM (5.5 mM glucose; Gibco, USA) supplemented with 10% FBS (Gibco, Grand Island, New York, USA) at 37°C and 5% CO2. The cell culture medium was refreshed every 2 days, and cells were passaged at 80–90% confluence. Upon reaching 80% confluence, the cells were seeded in 6-well plates (Labselect, Anhui, China). They were divided into different groups based on glucose concentration: normal glucose group (NG, 5.5 mM), high glucose groups (HG1, 35 mM; HG2, 55 mM; HG3, 75 mM; HG4, 95 mM), and mannitol group (5.5 mM glucose concentration and 49.5 mM mannitol in the culture medium) as an osmotic control group.
The hsa-miR-204-5p agomir and agomir negative controls (agomir-NC) were chemically synthesized by GenePharma (Shanghai, China). The agomir for overexpression consisted of a double strand with the following sequence: miR-204-5p agomir sense: 5'-UUCCCUUUGUCAUCCUAUGCCU-3' and antisense: 5'-GCAUAGGAUGACAAAGGGAAUU-3'; miR-204-5p agomir NC sense: 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense: 5'-ACGUGACACGUUCGGAGAATT-3'. HLECs were transfected with agomir or NC according to the manufacturer's instructions. Transfection was performed when the cells reached 60–80% confluence, using an advanced DNA RNA transfection reagent (#AD600025, Zeta Life, USA). The group that received only the transfection agents was referred to as the mock group. After 24 h of transfection, the culture medium was replaced with a new DMEM containing 55 mM glucose. The harvested cells were then analyzed in the subsequent assay at 48 h after transfection.
2. 3. RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)
Total RNA was extracted from collected HLECs and capsules, by EZB-RN001plus (EZBioscience, Roseville, MN, USA) according to the manufacturer’s protocol. After the concentration of RNA was determined by the Nanodrop 2000 system (Thermo, Boston, USA), 1 µg RNA was used to synthesize cDNA with UNI All-in-One SuperMix and gDNA remover (TransGen Biotech, Beijing, China). Detection of gene mRNA expression by SYBR Green qPCR SuperMix (TransGen Biotech, Beijing, China). To evaluate the expression levels of miR-204-5p, we synthesized the cDNA of miR-204-5p using specific reverse transcriptase primers (Genepharma, Shanghai, China). U6, GAPDH and β-actin serve as housekeeping genes for miRNA and mRNA detection, respectively. Relative miRNA and mRNA expression levels were determined according to the 2−ΔΔCt method. The sequences of the related primers were provided in Supplementary Table S1.
2. 4. Western Blotting
When HLECs confluence reached approximately 80%, total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (TransGen Biotech, Beijing, China) on ice. The protein concentration was measured using a BCA protein assay kit (Thermo, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to a 0.45 µm polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk in TBST at room temperature for 2 h and then incubated overnight at 4°C with primary antibodies against GAPDH (dilution 1:1000, Abcam, Cambridge, MA, USA) and TXNIP (dilution 1:1000; Huabio, Hangzhou, China). Subsequently, the membranes were incubated with secondary antibodies at room temperature for 1.5 h. Following the washing of the membranes, the protein bands were detected using ECL chemiluminescence (Beyotime, Shanghai, China). GAPDH was utilized as the internal control to determine the gray value ratio of each protein using ImageJ software (National Institutes of Health).
2. 5. Detection of Cellular ROS Level and Superoxide Dismutase (SOD) Activity
The endogenous ROS content of HLECs was detected using a ROS assay kit (Solarbio, Beijing, China), following the manufacturer's instructions. The fluorescence intensity of DCFH-DA was measured using a Cytation™ 5 Cell Imaging Multimode Reader (BioTek, VT, USA).
Total SOD activities of HLECs were measured by the total superoxide dismutase assay kit (S0101, Beyotime, Shanghai, China) with WST-8 according to the manufacturer's protocol. Microplate readers (BioTek, Vermont, USA) were used to detect absorbance in 450 nm.
2. 6. Cell Counting Kit-8 (CCK-8) Assay
Cell viability was assessed using CCK-8 assays (Beyotime, Shanghai, China). HLECs were seeded into a 96-well plate at a density of 1×10− 4 cells per well. Next, 100 µL of basic medium containing 10% CCK-8 reagent was added to each well, followed by incubation at 37°C in a light-protected environment for 2 h. Microplate readers (BioTek, Vermont, USA) were used to detect absorbance in 450 nm.
2. 7. Luciferase Reporter Assay
The putative binding sites of miR-204-5p and TXNIP were predicted using the miRNA target gene prediction tools, namely, Starbase (https://rnasysu.com/encori/), mirwalk (http://mirwalk.umm.uni-heidelberg.de), miRTarBase (https://mirtarbase.cuhk.edu.cn/), and miRPathDB (https://mpd.bioinf.uni-sb.de). The 3’ UTR sequences including wild-type (TXNIP-WT) or mutant-type binding sites (TXNIP-MUT) were constructed into pmirGlO luciferase reporter vector (GenePharma, Shanghai, China). The miR-204-5p agomir or NC were co-transfected with reporter plasmids into HLECs using Transfection Reagent. After 48 h of transfection, the cells were lysed according to the instructions of the dual-luciferase reporter assay kit (Beyotime, Shanghai, China), and the luciferase activity was measured using the Fluorescence Analyzer (BioTek, Vermont, USA).
2. 8. Apoptosis Assay
HLECs were transfected with the miR-204-5p agomir or agomir-NC for 24 h and then exposed to HG conditions for 24 h for the apoptosis assay. The Mitochondrial Membrane Potential (MMP) detection kit (JC-10, Zeta Life, USA) was used according to the manufacturer’s instructions. Using a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek, Vermont, USA) to measure the fluorescence intensity of JC-10.
Annexin V-FITC/propidium iodide (PI) kit (Solarbio, Beijing, China) was used to detect the apoptosis of HLECs. The cells were digested using EDTA-free trypsin, and the collected cells were resuspended in 100 µL of 1 x binding buffer. Subsequently, 5 µL of Annexin V-FITC was added and incubated for 5 minutes at room temperature in the dark, followed by the addition of 5 µL of PI and 400 µL of PBS. The Flow Cytometer (BD FACSAria III, USA) was utilized to determine the apoptosis rate in each group.
2. 9. Enzyme-linked Immunosorbent (ELISA) Assay
The levels of biologically active IL-1β content in cell culture supernatant were detected by a human IL-1β ELISA assay kit (Liankebio, Hangzhou, China) according to the protocol. Absorbance was measured using a microplate reader (BioTek, Vermont, USA) at a wavelength of 450 nm and a reference wavelength of 630 nm.
2. 10. Statistical Analysis
GraphPad Prism 9 (GraphPad, San Diego, CA, USA) was used for statistical analysis. Data were presented as the mean ± SEM. Differential expression was analyzed using an unpaired t-test between two groups and one-way ANOVA for multiple groups. Each experiment was independently repeated three times. Statistical significance was considered at P < 0.05.