Construction of a HSC activation-related lncRNA-miRNA-mRNA ceRNA regulatory network reveals potential molecules involved in liver fibrosis

Kaiqiang Wan^1,2Qiong Zhou^1,2Ruirui Feng^1,2Tao Liu^1,2Chang Fan^1,2Xue Pang^1,2Zhongliang Li^1,2Qiumei Zhou^1,2*

• ¹Anhui University of Chinese Medicine, Hefei, China
• ²The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, China

Background: Liver fibrosis (LF) represents a progressive pathophysiological consequence of persistent liver injury. While the competitive endogenous RNA (ceRNA) network serves as a critical regulator in diverse disease pathogenesis, its molecular underpinnings in LF and fibrogenic mediators remain unknown. Objective: In this study, we aimed to systematically probe the LF-related ceRNA regulatory axis, and identify the potential molecules involved in the Hepatic stellate cells (HSCs) activation. Methods and Results: Based on the whole transcriptome RNA sequencing, 401 lncRNAs, 60 miRNAs and 1224 mRNAs were identified between model and normal liver tissue samples. Then, through target gene prediction, a lncRNA-miRNA-mRNA (LMM) ceRNA network comprising 4 differentially expressed lncRNAs (DE lncRNAs), 6 DE miRNAs, and 148 DE mRNAs was established. The expression levels of these RNAs were verified by RT-qPCR. Functional annotation via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that target mRNAs of co-dysregulated lncRNAs and miRNAs in model groups were significantly enriched in multiple pathways, such as unsaturated fatty acids and TGF-β signaling pathways. Notably, 4 hub mRNAs (HMGCR, SREBF-1, TGF-β3 and FBN1) were identified by constructing Protein-protein interaction (PPI) network with the 148 DE mRNAs. Importantly, the dual-luciferase reporter assay confirmed the existence of specific binding sites among lncRNA H19, miR-148a-3p, and FBN1. Finally, the gene expression levels of were verified by RT-qPCR in TGF-β1-induced JS-1 cells, revealing that 5 lncRNA-miRNA-mRNA relationship pairs, containing H19, miR-130a-3p, miR-148a-3p, TGF-β3, FBN1 and HMGCR, were involved in the activation of HSCs. Conclusions: In this study, a HSC activation-related ceRNA network was successfully established in mice liver tissue, which could provides a novel framework for elucidating pathogenic mechanisms and identifying clinically relevant prognostic markers in LF progression.