A microRNA signature for valproate-induced steatosis in human hepatocytes and its application to predict fatty liver in paediatric epileptic patients on valproate therapy

Abstract

Valproate (VPA) has been the first-line, most frequently prescribed antiepileptic drug in children over the past 50 years. VPA causes, idiosyncratic hepatotoxicity in some patients, who often presents with hepatic steatosis. Experimental studies also support that VPA has high potential to induce steatosis. However, there is an apparent lack of significant hepatic problems in neuropediatric units, likely because iatrogenic liver steatosis lacks specific biomarkers. Thus, it is possible that a relevant number of children under VPA have asymptomatic fatty liver.

Aims

1) to demonstrate VPA-induced triglyceride (TG) accumulation in cultured human upcyte hepatocytes, 2) to identify miRNAs that are deregulated by VPA and associated with TG levels in these cells, and 3) to test these miRNAs, as potential non-invasive biomarkers, in plasma of paediatric epileptic patients on VPA, to identify those with a potential risk of liver steatosis.

Human upcyte hepatocytes were exposed to subcytotoxic VPA concentrations. Hepatocytes increased intracellular TGs by 27 % and 45 % after 2 and 4 mM VPA for 24 h. The profiling of cellular miRNAs by microarray analysis after 4 mM VPA identified 43 deregulated human miRNAs (fold-change > 1.5 or < −1.5; FDR p<0.05). Some of them (n=11), which were validated by RTqPCR and showed correlation (Pearson r≥ 0.6) with intracellular TG levels, were selected as potential VPA-induced steatosis biomarkers. Next, we investigated the expression of these miRNAs in human plasma and found that 9 of them could be reliably quantified by RTqPCR: miR-485-3p, miR-127-3p, miR-30a-3p, miR-92b-3p, miR-212-3p, miR-182-5p, miR-183-5p, miR-500a-5p and miR-675-5p. Screening of this 9-miRNA signature in 80 paediatric epileptic patients on VPA identified 18 patients (23 %) that clustered separately because of important alterations in the selected plasma miRNAs. These patients were younger and had higher VPA blood concentrations and serum liver enzyme levels.

In conclusion, VPA induced both TG accumulation and deregulation of a set of miRNAs in cultured human hepatocytes. Nine of these miRNAs have demonstrated potential as circulating biomarkers to identify VPA-induced steatosis in epileptic patients, which should require closer clinical follow-up.