Whole-transcriptome sequencing revealed differentially expressed mRNAs and non-coding RNAs played crucial roles in NiONPs-induced liver fibrosis

Abstract

Nickel oxide nanoparticles (NiONPs) induced liver fibrosis, while its mechanisms associated with transcriptome remained unclear. This study aimed to investigate the roles of differentially expressed (DE) messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) in NiONPs-induced liver fibrosis, and further confirm whether JNK/c-Jun pathway enriched by the DE RNAs was involved in the regulation of the disease. A liver fibrosis rat model was established by intratracheal perfusion of NiONPs twice a week for 9 weeks. Whole-transcriptome sequencing was applied to obtain expression profiles of mRNAs, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) in the model rat and control liver tissues. Comparing the RNA expression profiles of the model and control liver tissues, we identified 324 DE mRNAs, 129 DE lncRNAs, 24 DE miRNAs and 33 DE circRNAs, and the potential interactions among them were revealed by constructing two co-expression networks, including lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks. Using RT-qPCR, we verified the sequencing results of some RNAs in the networks and obtained similar expression profiles, indicating our sequencing results were reliable and referable. Through Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we predicted the biological functions and signaling pathways potentially related to NiONPs-induced liver fibrosis, such as "positive regulation of JNK cascade", "inflammatory response", "transcription factor binding", and MAPK, Wnt, PI3K-Akt signaling pathways. JNK/c-Jun pathway, a subclass of MAPK signal, was selected for further investigation because it was significantly enriched by fibrosis-related DE genes and activated in animal models. In vitro, we detected the cytotoxicity of NiONPs on LX-2 cells and treated the cells with 5 μg/ml NiONPs for 12 h. The results showed NiONPs induced the up-regulated protein expression of fibrotic factors collagen-1a1 (Col-1a1) and matrix metalloproteinas2 (MMP2) and JNK/c-Jun pathway activation. While these effects were reversed after JNK/c-Jun pathway was blocked by SP600125 (JNK pathway inhibitor), indicating the pathway was involved in NiONPs-induced excessive collagen formation. In conclusion, our results revealed the DE mRNAs and ncRNAs played crucial roles in NiONPs-induced liver fibrosis, and JNK/c-Jun pathway mediated the development of the disease.

Keywords: DE RNA; JNK/c-Jun pathway; Liver fibrosis; NiONPs; Whole-transcriptome sequencing.