Differential expression of microRNAs in bile duct obstruction-induced liver fibrosis and the identification of a novel liver fibrosis marker miR-1295b-3p.

Abstract

Bile duct obstruction-induced liver fibrosis is mainly caused by cholestatic liver injury which stimulates liver cell inflammation and damages the liver structure, causing liver fibrosis. The differentially expressed microRNAs and the potential target genes and signal pathways that are involved in bile duct obstruction-induced liver fibrosis remain unclear. We examined the differential expression of microRNAs and the target genes in the liver tissues of patients with liver fibrosis. High-throughput sequencing was used to detect the total microRNAs and identify the differentially expressed microRNAs. The topGO software was used to perform the Gene Ontology (GO) function enrichment analysis. The KOBAS software was used to analyze the associated biochemical metabolic pathways and signal transduction pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses were conducted to detect the expression of miR-1295b-3p, alpha smooth muscle actin (α-SMA), Bcl-2, caspase-3, Bax, and β-arrestin1 (ARRB1). Cell viability and apoptosis were detected by the Cell Counting Kit 8 (CCK-8) assay and flow cytometry. The targeting relationship between ARRB1 and miR-1295b-3p was verified using luciferase reporter assays. A total of 44 microRNAs were found to be differentially expressed, including 18 upregulated and 26 downregulated microRNAs. Five downregulated microRNAs, including miR-483-3p, miR-5589-3p, miR-1271-5p, miR-1295b-3p, and miR-7977. GO functional enrichment analysis of the target genes revealed the molecular functions, cellular location, and biological processes involved. Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway analysis showed that the target genes are mainly involved in metabolic pathways. In addition, the results of qRT-PCR revealed that miR-1295b-3p was downregulated in human fibrotic liver tissues and TGF-β1-activated LX-2 cells (human hepatic stellate cell line). Overexpression of miR-1295b-3p alleviated liver fibrosis, decreased the α-SMA levels, and inhibited proliferation and enhanced apoptosis in LX-2 cells. Dual-luciferase assays revealed that miR-1295b-3p suppressed ARRB1 expression by binding directly to its 3' untranslated region (UTR). This study identified the differentially expressed microRNAs in bile duct obstruction-induced liver fibrosis and revealed the potential target genes and signal pathways involved. Overexpression of miR-1295b-3p alleviated liver fibrosis, however, the specific targeting mechanisms warrant further clarification. Therefore, overexpressing miR-1295b-3p may be a potential treatment method for liver fibrosis.