Protective Role of microRNA-31 in Acetaminophen-Induced Liver Injury: A Negative Regulator of c-Jun N-Terminal Kinase (JNK) Signaling Pathway

Abstract

Background & Aims Sustained c-Jun N-terminal kinase (JNK) activation plays a major role in drug-induced liver injury (DILI). Stress-responsive microRNA-31 (miR-31) has been implicated in regulating different cellular damage, and JNK activation could induce miR-31 expression. However, the regulatory role of miR-31 in DILI has not been studied previously. We aimed to investigate whether miR-31 could ameliorate DILI and ascertain potential molecular mechanism. Methods miR-31 gene knockout (31-KO) and wild-type C57BL/6J mice were used to construct an acetaminophen (APAP)-induced DILI model. Primary mouse hepatocytes, as well as alpha mouse liver 12 (AML-12) cell lines, were used for in vitro experiments. Argonaute 2–associated RNA immunoprecipitation combined with high-throughput sequencing were performed to identify specific targets of miR-31. Results 31-KO mice showed a higher mortality rate, liver transaminase levels, and hepatic necrosis compared with those in wild-type mice after APAP-induced hepatotoxicity. The protective role of miR-31 on hepatocytes has been analyzed via constructing bone marrow chimeric mice. Mechanistically, we found that hepatic JNK phosphorylation increased significantly in 31-KO mice. This caused mitochondrial phosphorylated Src (p-Src) inactivation and more reactive oxygen species production, which directly amplifies hepatocyte necrotic cell death, while administration of JNK-specific inhibitor SP600125 could abrogate the differences. Moreover, bioinformatics analysis of RNA immunoprecipitation combined with high-throughput sequencing identified that guanosine triphosphatase, cell division cycle protein 42 (Cdc42), the upstream molecule of JNK signaling, was the specific target of miR-31 and could form a miR-31/Cdc42/phosphorylated mixed-lineage kinase 3 (p-MLK3) negative feedback loop to restrict JNK overactivation. Clinically, both miR-31 and phosphorylated JNK (p-JNK) were highly increased in liver tissues of DILI patients with different etiologies. Conclusions miR-31 can down-regulate Cdc42 to restrict overactivation of reactive oxygen species/JNK/mitochondria necrotic death loop in hepatocytes of APAP-induced DILI, which might provide a new therapeutic target for alleviating JNK overactivation–based liver injury.

BTF3L4 Overexpression Mediates APAP-induced Liver Injury in Mouse and Cellular Models

“…Damaged mitochondria cause liver injury by triggering an inflammatory cascade and generating excessive ROS. 43 Consistent with a previous study, 22 our findings demonstrate a significant increase in ROS levels in the AILI cellular model. Our investigation also revealed deterioration in mitochondrial morphology in hepatic cells subjected to APAP treatment, reflecting mitochondrial dysfunction to some degree.…”

“…For a cellular model of APAP-induced acute liver injury, alpha mouse liver 12 (AML-12) cells were treated with APAP at a final concentration of 30 µM (APAP in PBS) or vehicle (PBS) as previously described. 22 AML-12 cells were obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37 °C with 95% air and 5% CO 2 .…”

Genetic and Epigenetic Basis of Drug-Induced Liver Injury

“…Hence, it is important to understand the biogenesis, functions, and how miRNAs can act as biomarkers in DILI. 61…”

“…MiRNAs regulate and modulate the responses to xenobiotics in the liver. Hence, it is important to understand the biogenesis, functions, and how miRNAs can act as biomarkers in DILI 61 .…”

“…Hence, it is important to understand the biogenesis, functions, and how miRNAs can act as biomarkers in DILI. 61 MiRNAs regulating ROS: Amongst all miRNAs, the most liver-specific miRNAs which are found to be elevated in DILI are miRNA-122 and miRNA-192. 57,[62][63][64] However, Bakshi et al have shown that the levels of miR-122 and miR-192 are significantly reduced in DILI-AT.…”