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.2013 Sep;5(9):1402-14.
doi: 10.1002/emmm.201201900. Epub 2013 Jul 22.

MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction

Affiliations

MicroRNA expression profile and functional analysis reveal that miR-382 is a critical novel gene of alcohol addiction

Jingyuan Li et al. EMBO Mol Med.2013 Sep.

Abstract

Alcohol addiction is a major social and health concern. Here, we determined the expression profile of microRNAs (miRNAs) in the nucleus accumbens (NAc) of rats treated with alcohol. The results suggest that multiple miRNAs were aberrantly expressed in rat NAc after alcohol injection. Among them, miR-382 was down-regulated in alcohol-treated rats. In both cultured neuronal cells in vitro and in the NAc in vivo, we identified that the dopamine receptor D1 (Drd1) is a direct target gene of miR-382. Via this target gene, miR-382 strongly modulated the expression of DeltaFosB. Moreover, overexpression of miR-382 significantly attenuated alcohol-induced up-regulation of DRD1 and DeltaFosB, decreased voluntary intake of and preference for alcohol and inhibited the DRD1-induced action potential responses. The results indicated that miRNAs are involved in and may represent novel therapeutic targets for alcoholism.

Keywords: DeltaFosB; alcohol addiction; dopamine receptor D1; miR-382; microRNAs.

© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

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Figures

Figure 1
Figure 1. The expression of miR-382 in the nucleus accumbens (NAc) of rats after treatment with alcohol: 18 male rats were treated with vehicle (500 µl saline, i.p. bid) or alcohol (1 g/kg, i.p. bid)
Seven days later, the animals were sacrificed and their NAc were isolated for miRNA microarray analysis, and miR-382 measurement by qRT-PCR. ***Student'st-testp < 0.0001, compared with that in vehicle-treated controls. Values are mean ± SEM from nine rats.

  1. miR-382 was decreased in alcohol-treated rats as determined by microarray analysis.n = 9,p = 1.93E-10.

  2. miR-382 was decreased in alcohol-treated rats as determined by qRT-PCR.n = 9,p = 6.53E-9.

Figure 2
Figure 2. The effect of miR-382 on the expression of DRD1 and DeltaFosB in rat NAc: **p < 0.01, ***p < 0.001, Student'st-test
Source data is available for this figure in the Supporting Information.

  1. Seven-days' alcohol injection (1 g/kg, i.p. bid) significantly increased the expression of DRD1 (p = 0.00106) and DeltaFosB (p = 0.00056) in rat NAc at the protein level. Values are mean ± SEM from 6 independent experiments (n = 6), compared with that in vehicle-treated controls.

  2. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle or alcohol.

  3. Alcohol injections significantly increased the expression of DRD1 (p = 0.00022) and DeltaFosB (p = 0.00047) in rat NAc at the mRNA level. Values are mean ± SEM from 6 independent experiments (n = 6), compared with that in vehicle-treated controls.

  4. miR-382 expression in rat NAc was down-regulated by LNA-anti-miR-382 (Anti-miR-382) (p = 0.00133), but was up-regulated by Ad-miR-382 (p = 0.00102). Values are mean ± SEM from 4 independent experiments (n = 4), compared with that in the controls [LNA-anti-miR-382 control (Anti-control), and Ad-GFP].

  5. Left panel: view of injection site. Right panel: fluorescent image of GFP (green colour) in NAc at 5 days after microinjection of Ad-GFP. Injected Ad-GFP was limited to core region. Note: 40 µm slices, scale bar = 500 µm. aca, anterior commissure; AcBC, nucleus accumbens core; AabSH, nucleus accumbens shell; LV, lateral ventricle.

  6. The expression of DRD1 (p = 0.00057) and DeltaFosB (p = 0.0004) in rat NAc was increased by LNA-anti-miR-382. Values are mean ± SEM from 6 independent experiments (n = 6), compared with that in controls (Anti-control).

  7. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle, anti-control or Anti-miR-382.

  8. Overexpression of miR-382 via Ad-miR-382 decreased the expression of DRD1 (p = 0.00041) and DeltaFosB (p = 0.00087) in rat NAc. Values are mean ± SEM from 6 independent experiments (n = 6), compared with that in control (Ad-GFP).

  9. Representative Western blots of DRD1 and DeltaFosB from animals treated with vehicle, Ad-GFP or Ad-miR-382.

Figure 3
Figure 3.Drd1 is a direct target gene of miR-382 and is a regulator for the expression of DeltaFosB in cultured CAD cells: **p < 0.01, ***p < 0.001, Student'st-test
Source data is available for this figure in the Supporting Information.

  1. Drd1 is a potential target gene of miR-382 predicted by computational analysis.

  2. The luciferase reporter construct, containing the putative miR-382 binding sequence from 3′-UTR of ratDrd1 gene, was transfected into HEK293 cells with vehicle (Vehicle), an empty vector (pDNR-CMV), miR-382 (pmiR-31) or a control plasmid expressing an unrelated miRNA, miR-31 (pmiR-31). The construct with mutated fragment of the 3′-UTR ofDrd1 mRNA without the putative miR-382 binding sequences was used as the mutated control (mutatedDrd1), pmiR-382, but not pmiR-31 or pDNR-CMV, inhibited luciferase activity (p = 1.17571599413E-6). Values are mean ± SEM from 6 independent experiments (n = 6), compared with that in control (pDNR-CMV). In the mutated control group, the inhibitory effect of pmiR-miR-382 on luciferase activity disappeared. Values are mean ± SEM from 3 independent experiments (n = 3), compared with that in control (pDNR-CMV).

  3. The expression of miR-382 in cultured CAD cells was modulated by LNA-anti-miR-382 (Anti-miR-382) and pre-miR-382. Vehicle and control oligos (oligo control) were used as controls. miR-382 expression was down-regulated by LNA-anti-miR-382 (p = 5.08726615734E-5), but was up-regulated by pre-miR-382 (p = 1.69645597989E-5). Values are mean ± SEM from 5 independent experiments (n = 5), compared with that in control (oligo control).

  4. At protein level, pre-miR-382 decreased the expression of DRD1 (p = 2.44969469509E-5) and DeltaFosB (p = 0.0008). In contrast, the expression of DRD1 (p = 0.00089) and DeltaFosB (p = 0.00149) was increased by LNA-anti-miR-382 (Anti-miR-382). Values are mean ± SEM from 5 independent experiments (n = 5), compared with that in oligo control.

  5. Representative Western blots of DRD1 and DeltaFosB.

  6. pre-miR-382 decreased, whereas Anti-miR-382 increased the expression ofDrd1 (p = 0.00416 and 0.00039) andDeltaFosB (p = 7.21292762637E-5 and 0.00027) at mRNA level. Values are mean ± SEM from 5 independent experiments (n = 5), compared with that in oligo control.

  7. DRD1 protein was knocked-down by its siRNAs (DRD1-siRNA) (p = 0.00137). Values are mean ± SEM from 3 independent experiments (n = 3), compared with that in siRNA control.

  8. DeltaFosB protein was decreased via knocking-down of DRD1 by its siRNAs (p = 0.00817). Values are mean ± SEM from 3 independent experiments (n = 3), compared with that in siRNA control.

Figure 4
Figure 4. Overexpression of miR-382 is sufficient to inhibit alcohol-induced up-regulation of DRD1 and DeltaFosB in rat NAc: *p < 0.01, **p < 0.001, ##p < 0.001, Student'st-test
Source data is available for this figure in the Supporting Information. At 3 days before alcohol administration, 4 µl of vehicle, Ad-GFP or Ad-miR-382 (1 × 109 pfu/ml) was infused into the NAc of rats. Then, the animals were divided into the following groups: Vehicle-treated rats without alcohol administration (Vehicle); Ad-GFP-treated rats without alcohol administration (Ad-GFP); Ad-GFP-treated rats with alcohol administration (Alcohol + Ad-GFP) and Ad-miR-382-treated rats with alcohol administration (Alcohol + Ad-miR-382). Severn days later, the rat NAc were isolated.

  1. The successful modulation of miR-382 expression by Ad-miR-382. Alcohol decreased the expression of miR-382 in rat NAc (p = 0.00076). Values are mean ± SEM from 3 independent experiments (n = 3), Alcohol + Ad-GFP compared with that in Ad-GFP control. Ad-miR-382 increased the expression of miR-382 in NAc of rats with alcohol administration (p = 0.00079). Values are mean ± SEM from 3 independent experiments (n = 3), Alcohol + Ad-miR-382 compared with that in Alcohol + Ad-GFP control.

  2. Representative Western blots in rat NAc from different treatments.

  3. Downregulation of DRD1 and DeltaFosB via overexpression of miR-382 in NAc. Alcohol administration increased the expression of DRD1 (**p = 0.00616) and DeltaFosB (**p = 0.00087). Values are mean ± SEM from 5 independent experiments (n = 5), Alcohol + Ad-GFP compared with that in Ad-GFP control. Overexpression of miR-382 via Ad-miR-382 prevented alcohol-induced up-regulation of DRD1 (##p = 0.0002) and DeltaFosB (##p = 0.00012) in rat NAc. Values are mean ± SEM from 5 independent experiments (n = 5), Alcohol + Ad-miR-382 compared with that in Alcohol + Ad-GFP control.

Figure 5
Figure 5. Overexpression of miR-382 is sufficient to inhibit the voluntary intake of and the preference for alcohol in rats: the animals (totaln = 24) under the intermittent access two-bottle choice drinking paradigm were randomly divided into three groups which received infusion of Ad-miR-382, control adenovirus Ad-GFP or vehicle (saline), respectively, into the NAc
Seven days later, the rats were allowed to resume ethanol consumption under the same drinking paradigm. The successful up-regulation of miR-382 via Ad-miR-382 was verified by qRT-PCR at 7 days after drinking (Supporting Information Fig S1). The successful modulation of DRD1 and DeltaFosB via Ad-miR-382 was verified by Western blot analysis at 7 days after drinking (Supporting Information Fig S2).

  1. The voluntary intake of alcohol was reduced via Ad-miR-382 at all points (7 days:p = 0.00039; 9 days:p = 0.00046; 11 days:p = 0.00057; 13 days:p = 0.00026; 17 days:p = 0.00018; 19 days:p = 0.00098; 21 days:p = 0.013; 23 days:p = 0.0035).

  2. Ad-miR-382 decreased the preference for alcohol at all points (7 days:p = 0.0037; 9 days:p = 0.0041; 11 days:p = 0.0029; 13 days:p = 0.0015; 17 days:p = 0.0088; 19 days:p = 0.0068; 21 days:p = 0.018; 23 days:p = 0.0029).

  3. Ad-miR-382 did not alter the water consumption.

  4. Ad-miR-382 did not alter the total fluid intake. Values are mean ± SEM from 8 independent experiments (n = 8), compared with that in Ad-GFP control. *p < 0.05, **p < 0.01 ***andp < 0.001, two-way ANOVA with repeated measure.

Figure 6
Figure 6. Overexpression of miR-382 influences responses of MSNs in NAc slices to DRD1 activation: 4 µl of vehicle, Ad-GFP or Ad-miR-382 (1 × 109 pfu/ml) was infused into the NAc of rats
Severn days later, the NAc were isolated.

  1. A-C. The successful modulation of miR-382 expression by Ad-miR-382. Ad-miR-382 increased the expression of miR-382 in NAc of rats (p = 0.00125). Values are mean ± SEM from 3 independent experiments (n = 3), compared with that in Ad-GFP control. *p < 0.01, Student'st-test. Sample voltage traces in response to current injections (inset) from a MSN in acute brain slices of rats which received infusion of saline (B) and Ad-miR-382 (C), respectively. While the application of 1 µM SKF38393, the DRD1 agonist increased the firing rate of the MSN of rats that received saline injection, this was not seen in rats received Ad-miR-382 injection.

  2. D. Summary graphs showing that Ad-miR-382 NAc injection attenuated the firing rate of MSNs–induced by 1 µM SKF38393 (p = 0.00654). Values are mean ± SEM from 4 independent experiments (n = 4), compared with that in Ad-GFP control. **p < 0.01, two-way ANOVA.

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