High-Throughput Sequencing Reveals N6-Methyladenosine-modified LncRNAs as Potential Biomarkers in Mice with Liver Fibrosis

Abstract

Background: N⁶-methyladenosine (m⁶A) is the most frequent internal modification in eukaryotic RNA. Long noncoding RNAs (lncRNAs) are a new type of noncoding regulatory molecule with multiple cellular functions. Both are closely related to the occurrence and development of liver fibrosis (LF). However, the role of m⁶A-methylated lncRNAs in the progression of LF remains largely unknown.

Methods: In this study, HE and Masson staining were used to observe pathological changes in the liver, m⁶A-modified RNA immunoprecipitation sequencing (m⁶A-seq) was performed to systematically evaluate the m⁶A modification level of lncRNAs in LF mice, meRIP-qPCR and RT-qPCR were used to detect the m⁶A methylation level and RNA expression level of the target lncRNAs.

Results: A total of 415 m⁶A peaks were detected in 313 lncRNAs in liver fibrosis tissues. There were 98 significantly different m⁶A peaks in LF, which were located on 84 lncRNAs, of which 45.2% of the lncRNA length was between 200-400 bp. At the same time, the first three chromosomes of these methylated lncRNAs were chromosomes 7, 5 and 1. RNA sequencing identified 154 differentially expressed lncRNAs in LF. The joint analysis of m⁶A-seq and RNA-seq found that there were three lncRNAs with significant changes in m⁶A methylation and RNA expression levels: lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586. Subsequently, the verification results showed that the m⁶A methylation levels of lncRNA H19 and lncRNA Gm17586 were significantly increased, while that of lncRNA Gm16023 was significantly decreased, and the RNA expression of three lncRNAs was significantly decreased. Through the establishment of a lncRNA-miRNA-mRNA regulatory network, the possible regulatory relationships of lncRNA H19, lncRNA Gm16023 and lncRNA Gm17586 in LF were revealed.

Conclusion: This study revealed the unique m⁶A methylation pattern of lncRNAs in LF mice, suggesting that the m⁶A methylation modification of lncRNAs is related to the occurrence and development of LF.

Keywords: N6-methyladenosine; high-throughput sequencing; liver fibrosis; lncRNA; meRIP-qPCR; regulatory network.