Data Sheet 1_hsa_circ_0001377 promotes hepatic fibrosis by sponging let-7a-5p and targeting TGFBR1, Smad2, and Col1a2.doc

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.</p><p>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.</p><p>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.</p>