Oda Shingo, Hirabuki Yuka, Takeuchi Taiki, Kagawa Takumi, Yokoi Tsuyoshi
Department of Drug Safety Sciences, Division of Clinical Pharmacology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
J Appl Toxicol. 2021 Oct;41(10):1537-1552. doi: 10.1002/jat.4144. Epub 2021 Feb 9.
MicroRNAs (miRNA) have received considerable attention as potential biomarkers for drug-induced liver injury. We recently reported that the plasma levels of miR-143-3p and miR-218a-5p increased in severe cholestasis in rats. This study aimed to investigate whether these miRNAs increase in a severity-dependent manner and to elucidate their pathophysiological roles in cholestasis. Male Sprague-Dawley rats were orally administered different doses of α-naphthylisothiocyanate or 4,4-methylenedianiline to induce acute cholestasis. They were also orally administered acetaminophen or thioacetamide to induce hepatocellular injury. We found that plasma miR-143-3p and miR-218a-5p levels increased in a dose-dependent manner in cholestatic rats but not in hepatocellular injury. Bioinformatic analysis provided putative target genes of hsa-miR-218-5p, rno-miR-218a-5p, and mmu-miR-218-5p, among which GNAI2, PPP1CB, and PPP2R5A were experimentally validated as their direct target genes in human cholangiocyte line MMNK-1. Proliferation of MMNK-1 cells was significantly suppressed after overexpression of miR-218-5p and transduction of siRNAs for GNAI2, PPP1CB, and PPP2R5A. In the cholestatic livers of rats, Ppp1cb and Ppp2r5a expression levels decreased, whereas Gnai2 expression levels increased compared with those in vehicle-treated rats, suggesting that Ppp1cb and Ppp2r5a may be under the control of miR-218a-5p in vivo. In conclusion, our data suggest that miR-218(a)-5p is involved in the suppression of cholangiocyte proliferation by inhibiting the expression of PPP1CB and PPP2R5A, thereby contributing to the pathogenesis of cholestasis; and miR-218a-5p leaks into the plasma probably from damaged cholangiocytes in a severity-dependent manner in rats. Therefore, miR-218a-5p overexpression could be one of the underlying mechanisms of acute cholestatic liver injury in rats.
微小RNA（miRNA）作为药物性肝损伤的潜在生物标志物受到了广泛关注。我们最近报道，在大鼠严重胆汁淤积时，血浆中miR - 143 - 3p和miR - 218a - 5p水平升高。本研究旨在调查这些miRNA是否以严重程度依赖性方式升高，并阐明它们在胆汁淤积中的病理生理作用。雄性Sprague - Dawley大鼠经口给予不同剂量的α - 萘异硫氰酸酯或4,4 - 亚甲基二苯胺以诱导急性胆汁淤积。它们还经口给予对乙酰氨基酚或硫代乙酰胺以诱导肝细胞损伤。我们发现，胆汁淤积大鼠血浆中miR - 143 - 3p和miR - 218a - 5p水平呈剂量依赖性升高，但在肝细胞损伤中未升高。生物信息学分析提供了hsa - miR - 218 - 5p、rno - miR - 218a - 5p和mmu - miR - 218 - 5p的假定靶基因，其中GNAI2、PPP1CB和PPP2R5A在人胆管上皮细胞系MMNK - 1中经实验验证为其直接靶基因。miR - 218 - 5p过表达以及转导针对GNAI2、PPP1CB和PPP2R5A的小干扰RNA（siRNA）后，MMNK - 1细胞的增殖受到显著抑制。在大鼠胆汁淤积肝脏中，与给予溶媒的大鼠相比，Ppp1cb和Ppp2r5a表达水平降低，而Gnai2表达水平升高，这表明在体内Ppp1cb和Ppp2r5a可能受miR - 218a - 5p调控。总之,我们的数据表明，miR - 218(a) - 5p通过抑制PPP1CB和PPP2R5A的表达参与胆管上皮细胞增殖的抑制，从而促进胆汁淤积的发病机制；并且在大鼠中，miR - 218a - 5p可能以严重程度依赖性方式从受损胆管上皮细胞漏入血浆。因此，miR - 218a - 5p过表达可能是大鼠急性胆汁淤积性肝损伤的潜在机制之一。
