Lin, Chiu-HuaChiu-HuaLinYeh, Hsiang-YuanHsiang-YuanYehJao, Tzu-MingTzu-MingJaoCHIA-TER CHAOCHIH-KANG CHIANGJENQ-WEN HUANGKUAN-YU HUNG2026-02-112026-02-112026-02https://scholars.lib.ntu.edu.tw/handle/123456789/735924MicroRNAs (miRNAs) play crucial roles in the pathogenesis of vascular calcification (VC), with time-sensitive regulatory actions. However, few examine the temporal trajectories of miRNA expression during VC progression and their mechanistic implications. We integrated miRNomic datasets from vascular smooth muscle cells (VSMCs) and vascular tissues across consecutive time points for clustering-based trajectory analysis and miRNA-mRNA pairs dynamics. Key candidates were subjected to cross-model validation and functional assays. Six miRNAs (miR-202-3p, miR-186-5p, miR-221-3p, miR-222-3p, miR-92a-3p, and miR-30c-5p) displayed rising trajectories (RT), while 162 target mRNAs exhibited corresponding declining trajectories (DT). Conversely, two DT miRNAs (miR-143-3p and miR-93-5p) were matched with 24 RT mRNAs. Pathways such as stem cell pluripotency, TGF-β signaling, cellular senescence, and JAK-STAT were activated from early to mid-term VC and declined thereafter, whereas PIK/Akt, actin cytoskeleton regulation, and AGE/RAGE were successively upregulated. Experimental validation in in vitro and ex vivo VC models confirmed that miR-202-3p was an early responsive miRNA targeting BTG2, whose antagonism attenuated calcification. This study delineates the temporal landscape of miRNA and mRNA expression during VC. MiR-202-3p emerges as a potential early pro-calcific regulator and biomarker, whereas suppressing miR-202-3p may offer therapeutic efficacy. Our findings underscore the value of trajectory-based approaches in elucidating time-dependent VC drivers.enVascular calcificationmiRNomicmicroRNAjournal article10.1016/j.ijbiomac.2026.15044941577277