Chronic ethanol feeding enhances miR-21 induction during liver regeneration while inhibiting proliferation in rats

Abstract

Liver regeneration is an important repair response to liver injury. Chronic ethanol consumption inhibits and delays liver regeneration in experimental animals. We studied the effects of chronic ethanol treatment on messenger RNA (mRNA) and microRNA (miRNA) expression profiles during the first 24 h after two-thirds partial hepatectomy (PHx) and found an increase in hepatic miR-21 expression in both ethanol-fed and pair-fed control rats after PHx. We demonstrate that the increase of miR-21 expression during liver regeneration is more robust in ethanol-fed rats. Peak miR-21 expression occurs at 24 h after PHx in both ethanol-fed and control rats, corresponding to the peak of hepatocyte S phase in control rats, but not in ethanol-exposed livers in which cell cycle is delayed. The induction of miR-21 24 h after PHx in control rats is not greater than the increase in expression of miR-21 due to sham surgery. However, in the ethanol-fed rat, miR-21 is induced to a greater extent by PHx than by sham surgery. To elucidate the implications of increased miR-21 expression during liver regeneration, we employed unbiased global target analysis using gene expression data compiled by our group. Our analyses suggest that miR-21 may play a greater role in regulating gene expression during regeneration in the ethanol-fed rat than in the control rat. Our analysis of potential targets of miR-21 suggests that miR-21 affects a broad range of target processes and may have a widespread regulatory role under conditions of suppressed liver regeneration in ethanol-treated animals.

Fig. 1.

miR-21 expression during liver regeneration in chronically ethanol-fed (EtOH) and pair-fed control (CHO) rats. A: miR-21 expression was determined by RT-quantitative PCR in remnant livers during the first 36 h following partial hepatectomy (PHx). Each replicate was normalized to its own left lateral and medial lobes (LLM). At t = 0, EtOH LLM was normalized to CHO LLM and there was no significant difference in miR-21 expression between EtOH and CHO prior to PHx. Peak miR-21 expression was at 24 h after PHx in both EtOH and CHO rats. B: miR-21 levels are increased at 24 h after sham operation compared with the relative expression in the LLM tissue. Results found in chow-fed rats were comparable to results in CHO rats. In the EtOH rat, the increase in miR-21 24 h after PHx was greater than the increase in miR-21 due to sham operation. Data are mean relative expression ± SE, n = 4 for EtOH and CHO rats and n = 3 for chow-fed rats. For all symbols denoting significance, P < 0.05. *PHx or Sham vs. LLM; ^PHx vs. Sham; #EtOH PHx vs. CHO PHx.

Fig. 2.

Hepatocyte proliferation 24 h after PHx. A: labeling of bromodeoxyuridine (BrdU)-incorporated cells demonstrates that very few hepatocytes are replicating at 24 h after PHx in the EtOH rat. B: significantly fewer hepatocytes are replicating at 24 h after PHx in the EtOH rat (7.2%) compared with the CHO rat (35.8%). Data are means ± SE, n = 4. *P < 0.05.

Fig. 3.

Gene set enrichment analysis (GSEA) of miR-21-predicted targets after PHx. GSEA was performed for differentially expressed TargetScan-predicted miR-21 targets from 6 to 24 h after PHx in CHO (A) and EtOH (B). C: triple log₂ ratio (Δ2) of EtOH vs. CHO for differential expression from 6 to 24 h to demonstrate greater decrease in predicted targets over that time frame in EtOH than in CHO livers. GSEA-estimated FDR q values are reported; q < 0.05 is considered significant. D–F: corresponding cumulative distribution plots of differential expression of predicted miR-21 targets compared with differential expression of nontargets. P values were determined by Kolmogorov-Smirnov test. P < 0.05 is considered significant.

Fig. 4.

Gene ontology (GO) grouping of potential miR-21 targets identified by gene expression analysis by negative correlation. Identified genes have expression profiles that negatively correlate with miR-21 expression and are thus candidate targets for miR-21 in liver regeneration of ethanol-fed animals. Bar graphs represent differential expression at 6 and 24 h following PHx compared with LLM. All bar graphs are on a −1 to 1 log₂ scale with the following exceptions: Chd7, Dusp8, Nfib, PDCD4 are on a −2 to 2 log₂ scale and Btg2 and PPARα are on a −3.5 to 3.5 log₂ scale. *Potential regulation by miR-21 in CHO liver regeneration. §Genes that have previously been demonstrated to be miR-21 targets. †Pten is not predicted by TargetScan to be a target of miR-21.

Fig. 5.

miR-21 regulation of Crebl2, a predicted target identified by global target expression analysis. A: TargetScan-predicted miR-21–Crebl2 interaction. B: transfection of HEK293 cells with pre-miR-21 causes inhibition of a luciferase reporter gene linked to the 3′ untranslated region of Crebl2. Data are means ± SE, n = 3 experimental replicates of transfections with 3 technical triplicates per replicate. *P < 0.05.