Elucidating the regulatory role of Nrf2 in Beclin1-mediated autophagy in freshwater bivalve Hyriopsis cumingii

Abstract

Beclin1 is a central regulator of autophagy and cellular homeostasis, playing a critical role in the adaptation to environmental stress. Despite its importance, the expression patterns and regulatory mechanisms of Beclin1 in bivalves remain poorly understood. In this work, we cloned the Beclin1 gene from Hyriopsis cumingii (designated HcBeclin1) and investigated its regulatory interaction with nuclear factor erythroid 2-related factor 2 (HcNrf2). The full-length HcBeclin1 cDNA was 1365 bp and encoded a conserved open reading frame, sharing high sequence similarity with homologous genes in other species. Reverse transcription quantitative PCR analysis revealed that HcBeclin1 expression was significantly upregulated in the gills, hepatopancreas, and hemolymph in response to H₂O₂-induced oxidative stress. Notably, the expression levels of HcBeclin1 in the gills and hemolymph correlated positively with those of HcNrf2 under stress conditions. RNA interference experiments demonstrated that silencing HcNrf2 led to a marked downregulation of HcBeclin1, suggesting a regulatory relationship. The promoter region of HcBeclin1 was obtained through high-efficiency Thermal Asymmetric Interlaced PCR, revealing five putative HcNrf2 binding sites. Luciferase reporter assays identified a critical binding site within −937 to −663 bp region of the promoter, which was essential for transcriptional activation. Functional assays further confirmed that HcNrf2 regulated HcBeclin1 expression by binding to specific cis-acting elements within its promoter. These findings offer new insights into the molecular mechanisms underlying autophagy regulation in bivalves and highlight the pivotal role of HcBeclin1 in mediating cellular responses to oxidative stress.