Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression

Abstract

The expression of microRNA in nonalcoholic steatohepatitis (NASH) and their role in the genesis of NASH are not known. The aims of this study were to: (1) identify differentially expressed microRNAs in human NASH, (2) tabulate their potential targets, and (3) define the effect of a specific differentially expressed microRNA, miR-122, on its targets and compare these effects with the pattern of expression of these targets in human NASH. The expression of 474 human microRNAs was compared in subjects with the metabolic syndrome and NASH versus controls with normal liver histology. Differentially expressed microRNAs were identified by the muParaflo microRNA microarray assay and validated using quantitative real-time polymerase chain reaction (PCR). The effects of a specific differentially expressed miRNA (miR-122) on its predicted targets were assessed by silencing and overexpressing miR-122 in vitro. A total of 23 microRNAs were underexpressed or overexpressed. The predicted targets of these microRNAs are known to affect cell proliferation, protein translation, apoptosis, inflammation, oxidative stress, and metabolism. The miR-122 level was significantly decreased in subjects with NASH (63% by real-time PCR, P < 0.00001). Silencing miR-122 led to an initial increase in mRNA levels of these targets (P < 0.05 for all) followed by a decrease by 48 hours. This was accompanied by an increase in protein levels of these targets (P < 0.05 for all). Overexpression of miR-122 led to a significant decrease in protein levels of these targets.

Conclusions: NASH is associated with altered hepatic microRNA expression. Underexpression of miR-122 potentially contributes to altered lipid metabolism implicated in the pathogenesis of NASH.

Figure 1. Cluster analysis of differentially expressed miRNAs (p< 0.05)

miRNA signature in liver from patients with NASH and normal controls. Signal intensity was expressed as log₂ ratio between NASH and controls. Bright green, under-expression; black, no change; bright red, over-expression.

Figure 2. Quantitative RT-PCR validation of miRNA expression

The relative abundance of four specific miRNAs (25 NASH vs. 25 controls) was measured using RT-PCR and expressed as mean % change ± S.E.M. after normalization to GAPDH. miR-34a and miR-146b were confirmed to be overexpressed by 99% and 80% respectively while miR-122 was confirmed to be underexpressed by 63% (p< 0.02 for all). The expression of miR-451, which was not differentially expressed by microarray, was confirmed not to be significantly different between the two groups.

Figure 3. Multiplicity and cooperativity of miRNA-target interactions of differentially expressed miRNAs (p< 0.05)

One miRNA can target more than one gene (multiplicity) and one gene can be regulated by more than one miRNA (cooperativity). The distributions for multiplicity and cooperativity were based on (1) log₂ rank of differentially expressed miRNAs vs. number of gene targets (based on targets presented in tables S1-S6), and (2) log₂ rank of gene targets vs. number of miRNAs directed at them, respectively. Multiplicity and cooperativity of differentially over-expressed miRNAs were shown in panels a, c respectively, and differentially under-expressed miRNAs were shown in (b) and (d) respectively. Distributions decayed exponentially. Very few over- and under-expressed miRNAs regulated over 40 predicted targets (top left a-b), but most miRNAs controlled only a few genes (bottom right a-b). A few targets appeared to be under highly cooperative miRNA regulation (top left c-d), but most genes were targeted by fewer than four differentially expressed miRNAs (bottom right c-d).

Figure 4. Hepatic mRNA and protein expression of lipogenic genes in human subjects with NASH and matched controls

mRNA levels of hepatic lipogenic genes were expressed as relative fold change from a single human liver sample used as the internal calibrator across all RT-PCR experiments. Overall, the hepatic mRNA levels of FAS, SREBP-1c and HMGCR were increased significantly in subjects with NASH compared to controls (p< 0.05), while the hepatic mRNA level of SREBP-2 was not different between the two groups. As shown in panels b and c, SREBP-2, HMGCR, SREBP-1c and FAS protein were increased in NASH subjects (p< 0.02 for SREBP-2, p=ns for the rest).

Figure 5. Silencing of miR-122 in HepG2 cells

Using RT-PCR, mRNA expression of miR-122 targets involved in lipogenesis in subjects with NASH vs. controls was evaluated following silencing miR-122 in cell culture. As shown in a-d, mRNA levels of FAS, HMGCR, SREBP-1c and SREBP-2 increased significantly at 24hrs (p< 0.05 for all) after silencing of miR-122. The mRNA levels of these lipogenic genes then dropped significantly below baseline values by 48hrs (p< 0.02 for all). This was accompanied by a significant increase in protein expression of HMGCR, FAS and mature SREBP1c and SREBP2 (p< 0.02 for all) at 48hrs as shown in panels e and f (None: no treatment; scrambled: transfection with RNA duplex containing a scrambled sequence without known gene targets; anti: transfection with miR-122 silencing RNA).

Figure 6. Over-expression of miR-122 in HepG2 cells

Using RT-PCR, mRNA expression of miR-122 targets involved in lipogenesis was evaluated following over-expression of miR-122 in vitro. As shown in a-d, mRNA levels of FAS, HMGCR, SREBP-1c and SREBP-2 remained unchanged at 24hrs after over-expression. This was followed by a significant decrease in mRNA expression by 48hrs (p< 0.03 for all). Protein expression of HMGCR, FAS and mature SREBP1c and SREBP2 decreased significantly (p< 0.003 for all) at 48hrs after miR-122 over-expression as shown in panels e and f (None: no treatment; scrambled: transfection with RNA duplex containing a scrambled sequence without known gene targets; pre: transfection with miR-122 RNA).

Figure 7. Functional validation of miR-122 in Huh7 cells

Silencing of miR-122 resulted in a significant increase in HMGCR protein (p= 0.03), while over-expression of miR-122 led to decreased HMGCR protein expression.