Identification of Liver Fibrosis-Related MicroRNAs in Human Primary Hepatic Stellate Cells Using High-Throughput Sequencing

Abstract

MicroRNAs (miRNAs) participate in hepatic stellate cell (HSC) activation, which drives liver fibrosis initiation and progression. We aimed to identify novel hepatic fibrosis targets using miRNA sequencing (miRNA-seq) of human primary HSCs. Surgically resected liver tissues were used to extract HSCs. Based on next-generation sequencing, miRNA-seq was performed on four pairs of HSCs before and after in vitro culture. Additionally, we compared our data with open access miRNA-seq data derived from fourteen cirrhotic and nine healthy liver tissues. Selected miRNAs associated with fibrosis were verified by quantitative real-time PCR. Target mRNAs of differentially expressed (DE) miRNAs were predicted to construct co-expression networks. We identified 230 DEmiRNAs (118 upregulated and 112 downregulated) upon HSC activation. Of the 17 miRNAs with the most significant differences in expression, liver disease-related miRNAs included miR-758-3p, miR-493-5p, miR-409-3p, miR-31-5p, miR-1268a, and miR-381-3p, which might play roles in hepatic fibrosis. Moreover, let-7g-5p, miR-107, miR-122-5p, miR-127-3p, miR-139-5p, miR-148a-3p, miR-194-5p, miR-215-5p, miR-26a-5p, miR-340-5p, miR-451a, and miR-99a-5p were common between our data and the publicly available sequencing data. A co-expression network comprising 1891 matched miRNA–mRNA pairs representing 138 DEmiRNAs and 1414 DEmRNAs was constructed. MiR-1268a and miR-665, possessing the richest target DEmRNAs, may be vital in HSC activation. The targeted genes were involved in collagen metabolism, extracellular matrix structural constituent, cytoskeletal protein binding, and cell adhesion. The miRNAs we identified may provide a basis and reference for the selection of diagnostic and therapeutic targets for hepatic fibrosis.

Keywords

• human primary hepatic stellate cell
• liver fibrosis
• microRNA
• next-generation sequencing
• target