Long non-coding RNA LINC00657 aggravates atrial fibrosis in atrial fibrillation via the miR-144-3p/ROCK1 competing endogenous RNA axis.

The clinical significance and fibrogenic mechanisms of long non-coding RNA LINC00657 in atrial fibrillation (AF) remain unclear. This study aimed to elucidate whether LINC00657 served as a mechanistic driver of atrial fibrogenesis in AF via modulation of the microRNA-144-3p (miR-144-3p)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) axis. LINC00657 expression was quantified in peripheral blood from 150 AF patients and 150 matched controls. Angiotensin II (Ang II)-stimulated primary rat cardiac fibroblasts and continuously infused C57BL/6 mice served as in vitro and in vivo fibrosis models, respectively. Functional assays (cell counting kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blot) and mechanistic experiments (subcellular fractionation, RNA pull-down, RNA immunoprecipitation (RIP), dual-luciferase reporter) were conducted to dissect the LINC00657/miR-144-3p/ROCK1 competing endogenous RNA (ceRNA) axis. Clinically, elevated circulating LINC00657 discriminated AF patients from controls and independently predicted AF risk, correlating positively with high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity C-reactive protein (hs-CRP). In vitro, Ang II induced LINC00657 upregulation; silencing LINC00657 suppressed cardiac fibroblast proliferation, attenuated the release of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), reduced the expression of fibrotic markers including type I/III collagen and transforming growth factor-beta (TGF-β), and promoted apoptosis. In vivo, knockdown of LINC00657 significantly attenuated Ang II-mediated atrial fibrosis in mice. Electrocardiogram (ECG) monitoring and transesophageal atrial pacing further revealed that silencing LINC00657 significantly restored the atrial effective refractory period (AERP) and markedly decreased both the inducibility and duration of AF. Mechanistically, LINC00657 was predominantly localized to the cytoplasm and functioned as a ceRNA to sponge miR-144-3p, with ROCK1 identified as a direct downstream target of miR-144-3p. Both AF patients and model mice exhibited decreased miR-144-3p and increased ROCK1 expression, which negatively and positively correlated with LINC00657 levels, respectively. Rescue experiments demonstrated that miR-144-3p inhibitors partially reversed the anti-fibrotic effects of LINC00657 silencing, whereas further ROCK1 knockdown abrogated this reversal. Mechanistically, dissection of ROCK1 downstream signaling revealed that the LINC00657/miR-144-3p/ROCK1 axis specifically activated the ROCK1/myosin light chain 2 phosphorylation pathway, enhancing actomyosin contractility and stress fiber formation to drive the fibrotic cascade. The LINC00657/miR-144-3p/ROCK1 regulatory axis contributed to atrial fibrosis in AF.