Aging (Albany NY) · 7 authors, 2 centres
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This study demonstrates that microRNA-34a (MIR34A) is overexpressed in cataractous lenses and directly targets hexokinase 1 (HK1), suppressing its expression. Upregulation of MIR34A and downregulation of HK1 inhibit proliferation, induce apoptosis in lens epithelial cells, and accelerate lens opacification in mouse lenses via the HK1/caspase 3 signaling pathway. These findings provide a new molecular mechanism for cataract development and potential therapeutic targets.
**Background:** Cataracts are the leading cause of blindness worldwide, affecting 95 million people. Age is a major risk factor, and lens epithelial cell (LEC) abnormalities are central to cataract pathogenesis. MicroRNA-34a (MIR34A) is highly expressed in cataractous lenses, but its role in cataract development is unclear. Hexokinase 1 (HK1) is a rate-limiting enzyme in glycolysis, crucial for lens energy metabolism, as the lens is avascular and relies on anaerobic glycolysis. This study investigates whether MIR34A modulates LEC apoptosis and cataract formation by targeting HK1.
**Methods:** The study used the human LEC line SRA01/04 and mouse lens explants. MIR34A mimics, HK1 siRNA (siHK1), or deoxyglucose (HK1 inhibitor) were applied. Bioinformatics (MiRWalk, TargetScan) predicted HK1 as a MIR34A target, validated by dual-luciferase reporter assay in 293T cells. Gene expression was measured by RT-qPCR and western blot. Cell proliferation was assessed by CCK-8 assay at 48, 96, and 120 hours. Apoptosis was evaluated by flow cytometry (annexin V/PI staining) at 96, 120, and 144 hours and TUNEL assay at 120 hours. Mouse lens opacification was observed after 96 hours of treatment with Mir34a agomir, siHk1, or deoxyglucose. Immunofluorescence detected HK1 and active caspase 3 in cells and lens epithelium.
**Key Results:** MIR34A was overexpressed in cataractous lenses. MIR34A mimics significantly increased MIR34A expression (p<0.001) and reduced SRA01/04 cell proliferation at 48, 96, and 120 hours (p<0.05 to p<0.001). Flow cytometry showed increased apoptosis at 96, 120, and 144 hours (p<0.05 to p<0.001), and TUNEL confirmed more apoptotic cells at 120 hours. Luciferase reporter assay confirmed direct binding of MIR34A to the 3'UTR of HK1, with a significant decrease in luciferase activity for wild-type HK1 3'UTR (p<0.001) but not mutant. MIR34A overexpression suppressed HK1 mRNA and protein levels at 48, 96, and 120 hours (p<0.05 to p<0.001). siHK1 reduced HK1 mRNA and protein (p<0.001), inhibited proliferation at 48, 96, and 120 hours (p<0.05 to p<0.001), and increased apoptosis by flow cytometry at 96, 120, and 144 hours (p<0.05 to p<0.001) and TUNEL at 120 hours. In mouse lens explants, Mir34a, siHk1, and deoxyglucose each caused more severe opacification than control. Immunofluorescence showed decreased Hk1 expression in lens epithelium after Mir34a and siHk1 treatment, and increased TUNEL-positive cells. Active caspase 3 expression increased in SRA01/04 cells after MIR34A mimics or siHK1, and in lens epithelium after Mir34a, siHk1, or deoxyglucose treatment.
**Clinical Implications:** This study identifies a novel MIR34A-HK1-caspase 3 signaling pathway in cataract development. MIR34A overexpression in cataracts suppresses HK1, leading to LEC apoptosis and lens opacification. Targeting this pathway could offer new therapeutic strategies for cataract prevention or treatment, especially for age-related cataracts. However, the study is limited to in vitro and ex vivo models; in vivo validation is needed. Additionally, the role of HK2 and direct measurement of HK1 enzyme activity require further investigation. Despite these limitations, the findings provide a molecular basis for cataract pathogenesis and potential targets for intervention.