These authors share first authorship.
Version of Record online: 11 MAR 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 53, Issue 5, pages 1651–1661, May 2011
How to Cite
Sharma, A. D., Narain, N., Händel, E.-M., Iken, M., Singhal, N., Cathomen, T., Manns, M. P., Schöler, H. R., Ott, M. and Cantz, T. (2011), MicroRNA-221 regulates FAS-induced fulminant liver failure. Hepatology, 53: 1651–1661. doi: 10.1002/hep.24243
Parts of the study were funded by the German Research Foundation (Cluster of Excellence REBIRTH; EXC 62/1 awarded to Tobias Cantz and SFB 738 awarded to Michael Ott) and the German Ministry of Education and Research (ITCF-01GU0618, awarded to Toni Cathomen. Amar Deep Sharma receives grant support from HILF, Hannover Medical School.
Potential conflict of interest: Nothing to report.
- Issue online: 22 APR 2011
- Version of Record online: 11 MAR 2011
- Accepted manuscript online: 15 FEB 2011 12:15PM EST
- Manuscript Accepted: 2 FEB 2011
- Manuscript Received: 19 OCT 2010
Additional Supporting Information may be found in the online version of this article.
|HEP_24243_sm_SuppFig1.tif||1071K||Supporting Information Figure 1. Global loss of miRNAs by DROSHA silencing leads to increased apoptosis (a) Western blot demonstrates loss of DROSHA in Hepa 1-6 cells transduced with a retroviral vector expressing Drosha shRNA. (b) qRT-PCR shows efficient reduction of miR-122a, miR-221 and miR-21 in shDROSHA cells (Hepa 1-6 cells transduced with retroviral vector expressing shRNA against DROSHA) compared to control Hepa 1-6 cells. (c) Basal level of caspase-3/7 activity was similar in Hepa 1-6, shDGCR8 and shDROSHA cells without induction of apoptosis. Caspase activity of Hepa 1-6 was normalized to 100%. (d) WST assay revealed decreased cell survival in shDROSHA cells at 24 hr after in vitro apoptosis induction (e) Caspase-3/7 activity assay performed at 24 hr after Jo2-induced apoptosis detects increased apoptosis in shDROSHA cells. Error bars represent ± SEM. *P<0.05 and **P<0.005.|
|HEP_24243_sm_SuppFig2.tif||12989K||Supporting Information Figure 2. Identification of differentially regulated miRNAs in apoptotic liver (a) Increased serum ALT and AST levels at 6 hr and 12 hr after Jo2-induced acute apoptosis in the liver. (b) TUNEL staining confirmed progressive apoptosis in the liver at 6 hr and massive apoptosis at 12 hr after Jo2 injection with a dose of 0.4ug/gm body weight of mice. Original magnification x 200, Olympus IX71. (c) qRT-PCR revealed a similar expression pattern of miRNAs as observed in microRNA microarrays. (d) Efficient transfection of primary mouse hepatocytes with an Albumin-Gfp plasmid, which expresses Gfp under an albumin promoter (left) or cy-3 labeled control siRNA (right). Original magnification x 100, Olympus IX71. Error bars represents ± SEM. *P<0.05 and **P<0.005.|
|HEP_24243_sm_SuppFig3.tif||1016K||Supporting Information Figure 3. (a) PUMA protein levels in hepatocytes decreased at the time of massive apoptosis at 12 hr although transiently increased levels were seen at earlier time points after Jo2 injection in BALB/c mice. (b) Decreased luciferase activity in mouse hepatocytes transfected with miR-221 mimics and miR-GLO-PUMA. (c) Increased luciferase activity in mouse hepatocytes transfected with inhibitors of miR-221 and miR-GLO-PUMA. (d) Knockdown of PUMA by two different siRNA (1 or 2). Error bars represent ± SEM. **P<0.005.|
|HEP_24243_sm_SuppFig4.tif||1136K||Supporting Information Figure 4. (a) and (b)Western blot shows decreased PTEN and BMF protein levels in mouse hepatocytes at 48 hr after transfection with Pten siRNA (a) and Bmf siRNA (b) respectively. (c-e) miRNA target protectors for Puma, Bmf and Pten were transfected in primary hepatocytes. 48 hr after transfection western blots show the restoration of endogenous levels of PUMA (c), BMF (d) and PTEN (e). (f-g) miR-221 mimic or control mimic (100nM) were transfected in primary hepatocytes. Transfected hepatocytes were treated with TNF-α (25ng/ml). 24 hours after induction of apoptosis WST assay (f) and caspase-3/7 activity assay (g) revealed a modest miR-221 mediated protection against apoptosis. *P<0.05.|
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