MicroRNA expression profiling involved in MC-LR-induced hepatotoxicity using high-throughput sequencing analysis

ABSTRACT

Microcystin-LR (MC-LR), the most common microcystin (MC) present in water is known to pose a significant threat to human health especially hepatotoxicity. However, the specific molecular mechanisms underlying MC-LR-induced hepatic cellular damage still remain to be determined. MicroRNAs (miRNAs) are known to play key roles in cellular processes including development, cell proliferation and responsiveness to stress. Thus, this study aimed to examine, whether miRNAs were involved in the observed MC-LR-mediated liver damage using miRNA profiling of a human normal liver cell line HL7702 using high-throughput sequencing techniques. Protein phosphatase 2A (PP2A) activity, an established biomarker of microcystin toxicity, was determined 24 hr following treatment with the algal toxin to confirm responsiveness. Data demonstrated that MC-LR significantly inhibited PP2A activity in a concentration-dependent manner with inhibitory concentration (IC₅₀) value of 4.6 μM. Compared with control cells, treatment with MC-LR at concentrations of 1, 2.5, 5 or 10 μM significantly modified expression of levels of 3, 10, 9, and 99 miRNAs, respectively. Expression levels of miR-15b-3p were significantly increased in all 4 treatment groups, while miR-4521 expression levels were markedly reduced. In the case of miR-451a, 1, 5 or 10 μM also significantly lowered expression levels. However, a significant rise in miR-451a was noted in cells exposed to 2.5 μM toxin. The results obtained from miRNA differential expression levels were confirmed by real-time fluorescent quantitative PCR (qPCR). Gene Ontology (GO) enrichment analysis of hepatic cells demonstrated that miRNAs significantly altered were involved in systems development, metabolism, and protein binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis data showed that target genes of differentially expressed miRNAs in liver cells predominantly participated in mechanistic target of rapamycin kinase (mTOR), Ras, Ras-related protein 1 (Rap1), hypoxia inducible factor 1 (HIF-1), and cancer development. In summary, evidence indicates that MC-LR-induced hepatotoxicity may be associated with alterations in miRNAs. Evidence indicates that alterations in miR-451a, miR-4521 and miR-15b-3p may be involved in the observed MC-LR- induced hepatotoxicity

Acknowledgments

The authors are grateful to Professor Sam Kacew from Canada for revising and editing paper, and Dr. Chunlin Ou in the Central South Uinversity for designing experiments.

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Additional information

Funding

This study was supported by the National Natural Science Foundation (81502787, 81773393, 81472972); Hunan Province Natural Science Foundation (2016JJ3166); Chinese Postdoctoral Science Special Foundation (2016T90766), and Chinese Postdoctoral Science General Financial Foundation (2015M572273), the open-end fund for the valuable and precision instruments of Central South University (CSUZC201736), National Science and Technology Basic Project of the Ministry of Science and Technology of China (2015FY111100), and Open Funding (2014EME001).