Abstract
Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3′untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.
Highlights
The molecular pathogenesis of the highly prevalent chronic liver disease, nonalcoholic fatty liver disease (NAFLD) is not fully understood [1,2]
MicroRNAs decreased by palmitate treatment Due to our interest in lipotoxicity and endoplasmic reticulum (ER) stress, we utilized previously described mouse hepatocytederived cell lines to identify microRNAs that were downregulated by palmitate treatment and tunicamycin-induced ER stress [21]
We first identified microRNAs downregulated by palmitate (400 mM, 16 hours) or tunicamycin (1 mg/mL, 16 hours) in IRE1a wild-type hepatocyte cells (IRE-WT) and IRE1a knockout (IRE-KO) hepatocytes by pair-wise comparisons of microRNAs downregulated under these conditions, compared to vehicle treated cells (Data S1-S4)
Summary
The molecular pathogenesis of the highly prevalent chronic liver disease, nonalcoholic fatty liver disease (NAFLD) is not fully understood [1,2]. Progressive forms of NAFLD, termed nonalcoholic steatohepatitis (NASH) are characterized by hepatocyte apoptosis, which correlates with disease severity as well as disease progression to cirrhosis [3]. Circulating free fatty acids (FFA) are elevated in NASH, and when elevated induce apoptosis of cells, a process termed lipoapoptosis [4,5]. Recent studies have linked endoplasmic reticulum (ER) stress and microRNAs (miRs) to NAFLD. MicroRNAs are small noncoding RNAs increasingly recognized in modulating the cellular response to stress [6]. By posttranscriptionally regulating the expression of their target proteins, microRNAs are able to fine tune cellular protein levels and a cell’s response to stress. Recent studies have linked microRNAs to ER stress pathways; the role of microRNAs in regulating ER stress-induced cell death under lipotoxic conditions has not been explored
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