hsa_circ_0001377 promotes hepatic fibrosis by sponging let-7a-5p and targeting TGFBR1, Smad2, and Col1a2.

Background: Hepatic fibrosis (HF) is primarily driven by the activation of hepatic stellate cells (HSCs) and is characterized by excessive extracellular matrix (ECM) accumulation. Circular RNAs (circRNAs) have recently gained increasing attention in HF research. Methods: RNA sequencing and bioinformatic analyses revealed elevated hsa_circ_0001377 expression in human HF tissues. Its circular structure and subcellular localization were identified using Sanger sequencing, RNase R treatment, and fluorescence in situ hybridization (FISH). Gain- and loss-of-function experiments were conducted to elucidate the role of hsa_circ_0001377 in the human HSC line LX-2. Furthermore, the in vitro and in vivo effects of the hsa_circ_0001377/let-7a-5p/TGFBR1, Smad2, and Col1a2 regulatory axis were investigated using cytological assays and a bile duct ligation (BDL)-induced mouse model of HF. Results: hsa_circ_0001377 was significantly upregulated in the circRNA expression profile of human HF tissues. Its circular structure was validated through Sanger sequencing and resistance to RNase R digestion. FISH and co-staining with α-SMA demonstrated that hsa_circ_0001377 was abundantly expressed in activated HSCs (aHSCs; α-SMA-positive cells). Gain- and loss-of-function assays were performed using LX-2 cells with stable hsa_circ_0001377 overexpression and a specific siRNA targeting this circRNA. hsa_circ_0001377 overexpression promoted LX-2 cell activation, proliferation, migration, and G1/S-phase transition, whereas its knockdown produced the opposite effects. qRT-PCR, AGO-RIP, dual-luciferase reporter, and miRNA pulldown assays confirmed that hsa_circ_0001377 acts as a molecular sponge for let-7a-5p and that let-7a-5p targets TGFBR1, Smad2, and Col1a2. Rescue experiments in LX-2 cells further demonstrated that hsa_circ_0001377 promotes HSC activation, proliferation, migration, and G1/S-phase transition through the let-7a-5p/TGFBR1, Smad2, and Col1a2 axis. Moreover, hsa_circ_0001377 was highly expressed in 20 human HF samples, and its expression increased with advancing fibrosis stage. In vivo, a BDL-induced murine HF model demonstrated that the homologous mmu_circ_0001377 was also significantly upregulated in fibrotic liver tissues. In addition, tail-vein injection of an mmu-let-7a-5p agomir into BDL-induced HF mice confirmed the inhibitory effects of the let-7a-5p/TGFBR1, Smad2, and Col1a2 axis on α-SMA expression and collagen deposition. Conclusion: Taken together, these findings suggest that the hsa_circ_0001377/let-7a-5p/TGFBR1, Smad2, and Col1a2 regulatory axis may represent a promising therapeutic target for HF.