7 research outputs found

    Mmu-miR-615-3p Regulates Lipoapoptosis by Inhibiting C/EBP Homologous Protein

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    <div><p>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 <i>in silico</i> analyses, respectively. There is a single miR-615-3p binding site in the 3′untranslated region (UTR) of the <i>Chop</i> 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.</p></div

    MiR-615-3p inhibits CHOP expression.

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    <p>(A) Representative western blot for CHOP in IRE-WT cells treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin (tuni) for 16 hours. The cells were transfected with either a negative control, or a precursor of miR-615-3p (pre-miR-615-3p). Molecular weights are indicated in kDa. The immune complexes were detected using an infrared fluorescent imaging system. Alpha-tubulin was used as loading control. (B) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as A, expressed relative to the negative control mimic treated cells for each condition, respectively. ** P<0.01. (C) Representative western blot for CHOP in Hepa 1–6 cells treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 24 hours. The cells were transfected with either a negative control, or a precursor of miR-615-3p (pre-miR-615-3p). Molecular weights are indicated in kDa. The immune complexes were detected using enhanced chemiluminescence. Alpha-tubulin was used as loading control. The middle panel (**) depicts a longer film exposure of the top panel (*) (D) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as C, expressed relative to the negative control mimic treated cells for each condition, respectively. * P<0.05, ** P<0.01 (E) Representative western blot for CHOP in IRE-WT and Hepa1-6 cells in cells transfected with either an antagomir to miR-615-p or a negative control antagomir. Alpha-tubulin was used as loading control. (F) Quantification of CHOP protein levels normalized to tubulin, under the same conditions as E, expressed relative to the negative control antagomir treated cells for each condition, respectively. P = ns.</p

    <i>Chop</i> mRNA is a direct target of miR-615-3p.

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    <p>A region of the <i>Chop</i> 3′UTR containing the putative miR-615-3p binding site was cloned into the pMIR-report vector downstream of the luciferase coding region (p-MIR-Chop). The 3′UTR segment with the putative miR-615-3p binding site mutated (p-MIR-Chop-mut) was also cloned into the pMIR-report vector. HEK293 cells were co-transfected with the respective reporter plasmid and precursor of miR-615-3p (pre-miR-615-3p) or a negative control precursor molecule. Relative luciferase activity (normalized to renilla) was measured 24 hours after the transfection. Data are expressed relative to the wild-type binding site transfected cells treated with a negative control precursor molecule and (n = 5 independent experiments), * P<0.05.</p

    Palmitate treatment decreases miR-615-3p levels.

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    <p>(A) Neonatal IRE1α wild-type hepatocyte cells (IRE-WT) were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin (tuni) for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (B) Neonatal IRE1α knockout (IRE-KO) cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC (C) Primary mouse hepatocytes (PMH) were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 16 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (D) Hepa1-6 cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 24 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC. (E) Huh7 cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 30 hours. Control cells were treated with vehicle alone (VC). * P<0.05 compared to VC.</p

    MiR-615-3p reduces palmitate-induced cell death.

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    <p>(A) Neonatal liver wild-type cells were transfected with either the negative control precursor or precursor of miR-615-3p, both fluorescently labeled with Cy3. 8 hours after transfection, cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 18 hours. DAPI stained nuclei were counted in each condition. * P<0.05. (B) Neonatal liver wild-type cells were transfected with either the negative control precursor or precursor of miR-615-3p. 8 hours after transfection, cells were treated with 400 µM palmitate (PA) or 1 µg/mL tunicamycin for 18 hours. Caspase 3/7 activity was measured as described. * P<0.05.</p

    MicroRNAs downregulated by palmitate and tunicamycin treatment in IRE1α wild-type and knockout cells.

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    <p>MicroRNAs downregulated by palmitate and tunicamycin treatment in IRE1α wild-type and knockout cells.</p

    MiR-615-3p and CHOP levels in nonalcoholic steatohepatitis.

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    <p>(A) MiR-615-3p levels were measured in mouse liver from chow fed mice (n = 7) and mice with nonalcoholic steatohepatitis induced by feeding a diet high in fructose, fat and cholesterol (FFC) for 6 months (n = 13). Bars depict mean ±S.E.M, * <i>P</i><0.05. (B) Immunoblots for p-eIF2α, total-eIF2 α, ATF4, CHOP and GAPDH for loading control from whole liver protein extracts from chow fed mice (n = 3) and FFC-fed mice (n = ). The arrow points to the predicted CHOP band in the immunoblot.</p
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