Chinese Medical Journal · 6 authors, 2 centres
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This study demonstrates that lncRNA TUG1 is upregulated in age-related cataract (ARC) tissues and promotes lens epithelial cell apoptosis by acting as a competing endogenous RNA (ceRNA) that sponges miR-29b, thereby increasing Smac/DIABLO expression. The findings identify the TUG1/miR-29b/Smac axis as a novel molecular pathway in ARC development, offering potential non-surgical therapeutic targets for cataract treatment.
**Background:** Cataracts are a leading cause of blindness worldwide, with age-related cataracts (ARC) being the most common form in China. Currently, surgery is the only effective treatment, but it is costly and associated with complications. Apoptosis of lens epithelial cells (LECs) is considered the cytological basis for ARC formation. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are increasingly recognized as regulators of gene expression in various diseases. lncRNA TUG1 (taurine upregulation gene 1) is known to be involved in tumor biology, but its role in cataracts is poorly understood. Smac/DIABLO is a pro-apoptotic protein released from mitochondria during apoptosis. This study aimed to investigate whether TUG1 regulates LEC apoptosis in ARC by acting as a competing endogenous RNA (ceRNA) for miR-29b, thereby modulating Smac expression.
**Methods:** Anterior lens capsule samples were collected from 30 ARC patients (10 each with cortical, nuclear, and posterior subcapsular cataracts; LOCS III grade ≥4) and 10 control patients undergoing vitrectomy (opacity ≤2). Exclusion criteria included high myopia, uveitis, trauma, glaucoma, diabetic retinopathy, and systemic diseases. The human lens epithelial cell line HLE-B3 was used for in vitro experiments. An oxidative stress model was established by treating cells with 200 μmol/L H2O2 for 24 h (IC50 determined by CCK-8 assay). mRNA expression of TUG1, miR-29b, and Smac was measured by RT-qPCR in tissues and cells. Smac protein expression was analyzed by Western blotting and immunofluorescence. Cell apoptosis was assessed by flow cytometry (Annexin V-PE/7-AAD) and cell viability by CCK-8. Subcellular localization of TUG1 was determined by nucleocytoplasmic separation and RT-qPCR. The binding between TUG1 and miR-29b, and between miR-29b and Smac 3′-UTR, was predicted using TargetScan and Starbase, and validated by dual-luciferase reporter assays and RNA immunoprecipitation (RIP) with anti-Ago2 antibodies. Lentiviral vectors (sh-TUG1, pcDNA-TUG1) and miRNA mimics/inhibitors were used for gain- and loss-of-function studies. Statistical analysis used Student's t-test or one-way ANOVA; P<0.05 was considered significant.
**Key Results:** In ARC anterior capsule tissues, TUG1 and Smac mRNA were significantly higher than controls (both P<0.001), while miR-29b was lower (P<0.0001). In H2O2-treated HLE-B3 cells (200 μmol/L, 24 h), TUG1 and Smac were upregulated (TUG1: t=5.230, P=0.0005; Smac protein 1.5-fold increase, t=3.524, P=0.0244), and miR-29b was decreased (t=6.348, P<0.0001). Knockdown of TUG1 (sh-TUG1) significantly reduced TUG1 expression (t=14.940, P=0.0001), increased miR-29b (t=5.227, P=0.0004), decreased Smac protein (t=4.228, P=0.0134), and reduced apoptosis rate (t=3.008, P=0.0396). Subcellular fractionation showed 66.8% of TUG1 localized in the cytoplasm. Luciferase assays confirmed that miR-29b binds to TUG1 (TUG1-WT: t=4.504, P=0.0108; TUG1-Mut: P=0.6646) and to Smac 3′-UTR (Smac-WT: t=17.15, P<0.001; Smac-Mut: P=0.597). RIP experiments showed enrichment of TUG1 with Ago2 in miR-29b-overexpressing cells (t=8.696, P<0.01) and decreased enrichment with anti-miR-29b (t=7.663, P<0.01). Overexpression of miR-29b reduced Smac protein (t=9.942, P=0.0006) and apoptosis (t=25.340, P<0.01), while miR-29b inhibitor increased apoptosis (t=15.990, P<0.01). Rescue experiments showed that co-transfection of miR-29b mimic with pcDNA-TUG1 partially reversed the TUG1-induced increase in Smac protein (t=5.208, P=0.0065) and apoptosis (t=6.839, P=0.0024) compared to pcDNA-TUG1 alone.
**Clinical Implications:** This study identifies a novel molecular mechanism in ARC pathogenesis: lncRNA TUG1 acts as a ceRNA to sponge miR-29b, thereby upregulating Smac expression and promoting LEC apoptosis. The TUG1/miR-29b/Smac axis may serve as a potential therapeutic target for non-surgical intervention in age-related cataracts. Targeting this pathway could delay cataract progression and reduce the need for surgery, improving patient outcomes and reducing healthcare costs. However, these findings are based on in vitro and ex vivo experiments; further in vivo studies and clinical trials are needed to validate the therapeutic potential.