METTL14-mediated upregulation of lncRNA HOTAIR represses PP1α expression by promoting H3K4me1 demethylation in oxycodone-treated mice

Abstract

N6-methyladenosine (m6A) methylation is a vital epigenetic mechanism associated with drug addiction. However, the relationship between m6A modification and oxycodone rewarding is less well explored. Based on an open field test, the present study evaluated oxycodone rewarding using chromatin immunoprecipitation PCR, immunofluorescence, and RNA sequencing. A marked increase in METTL14 protein and a decrease in PP1α protein due to oxycodone abundance in the striatal neurons were observed in a dose- and time-dependent manner. Oxycodone markedly increased LSD1 expression, and decreased H3K4me1 expression in the striatum. In the open field test, intra-striatal injection of METTL14 siRNA, HOTAIR siRNA, or LSD1 shRNA blocked oxycodone-induced increase in locomotor activity. The downregulation of PP1α was also inhibited after treatment with METTL14/HOTAIR siRNA and LSD1 shRNA. Enhanced binding of LSD1 with CoRest and of CoRest with the PP1α gene induced by oxycodone was also reversed by LSD1 shRNA. In addition, H3K4me1 demethylation was also blocked by the treatment. In summary, the investigation confirmed that METTL14-mediated upregulation of HOTAIR resulted in the repression of PP1α, which in turn facilitated the recruitment of LSD1, thus catalyzing H3K4me1 demethylation and promoting oxycodone addiction.

Keywords: H3K4me1; LSD1; METTL14; m6A; oxycodone.

FIGURE 1

The experimental design of the present study.

FIGURE 2

Oxycodone altered the expression of mRNAs, ncRNAs, and lncRNAs in the striatum. (A) Volcanic plot of differentially expressed genes. The X coordinate is log₂ (fold change) and the Y coordinate is log₁₀ (Q‐value). Each point stands for a gene. Red points represent significantly upregulated genes. Green points represent significantly downregulated genes. Gray points represent genes with nonsignificant expression. (B) Representation of the 20 cellular components with the most significant differential gene enrichment. The X coordinate presents the rich ratio and the Y coordinate presents the cellular component terms. (C) Bubble plot of KEGG enrichment analysis. The X coordinate is the enrichment ratio of differentially expressed genes and the Y coordinate is the pathway.

FIGURE 3

Oxycodone downregulated striatal PP1α underlying m6A methylation. (A) Oxycodone (1.5, 3.0, and 6.0 mg/kg) was injected to all test animals. (B–D) Western blotting shows a significant increase in METTL14 expression and a decrease in PP1α expression. (E) Oxycodone markedly increased the movement of mice. *p < 0.05, **p < 0.01, ***p < 0.001, vs. baseline (day 1), two‐way ANOVA. (F–H) Western blotting shows the expressions of METTL14 and PP1α in striatal neurons. *p < 0.05, **p < 0.01, ***p < 0.001, vs. SAL group or day 1, one‐way ANOVA. Representative images of double immunofluorescence staining (I) of METTL14 or PP1α (red) and NeuN (green). METTL14 (J), m6A (L), and HOTAIR (M) levels were upregulated, while PP1α level (K) was downregulated in oxycodone‐treated mice. Scale bar (E) = 20 μm. *p < 0.05, **p < 0.01, ***p < 0.001, vs. SAL group or day 1, one‐way ANOVA.

FIGURE 4

Blockade of METTL14 lowered locomotor activity in oxycodone‐treated mice. (A) Expression of METTL14 in the primary cells was inhibited by METTL14 siRNA treatment. METTL14 siRNA1 (si1) was chosen because of its high silencing efficiency. **p < 0.01, ***p < 0.001, vs. scrambled nontargeting oligo (sc group), one‐way ANOVA. (B) Injection of METTL14 siRNA significantly decreased the movement of mice. **p < 0.01, ***p < 0.001, vs. SAL+Veh group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, vs. OXY + Veh group, two‐way ANOVA. (C) Significantly lesser movement was observed on days 11–12. METTL14 siRNA treatment markedly increased the expression of PP1α (D) while decreasing m6A (E) and HOTAIR levels (F). ***p < 0.001, vs. SAL+Veh group; ^## p < 0.01, ^### p < 0.001, vs. OXY + Veh group, one‐way ANOVA.

FIGURE 5

Blockade of HOTAIR lowered locomotor activity in oxycodone‐treated mice. (A) Expression of HOTAIR in the primary cells was inhibited by HOTAIR siRNA treatment. HOTAIR siRNA3 (si3) was chosen because of its high‐silencing efficiency. *p < 0.05, ***p < 0.001, vs. sc group, one‐way ANOVA. (B) Injection of HOTAIR siRNA significantly lowered the movement of oxycodone‐treated mice. **p < 0.01, ***p < 0.001, vs. SAL+Veh group; ^## p < 0.01, ^### p < 0.001, vs. OXY + Veh group, two‐way ANOVA. (C) Significantly lesser movement was observed on days 11–12. (D) HOTAIR siRNA treatment markedly increased the expression of PP1α in oxycodone‐treated mice. ***p < 0.001, vs. SAL+Veh group; ^### p < 0.001, vs. OXY + Veh group, one‐way ANOVA.

FIGURE 6

Treatment with AVV‐LSD1 shRNA inhibited locomotor activity in oxycodone‐treated mice. (A) Injecting AVV‐LSD1 shRNA 3 weeks prior to the open field test markedly decreased the locomotor activity of mice. **p < 0.01, ***p < 0.001, vs. SAL+Veh group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, vs. OXY + Veh group, two‐way ANOVA. (B) Fluorescence localization of AVV in the striatum. (C) Western blot showing increased expression of PP1α after LSD1 shRNA administration in oxycodone‐treated mice. ***p < 0.001, vs. SAL+Veh group; ^### p < 0.001, vs. OXY + Veh group, one‐way ANOVA.

FIGURE 7

LSD1‐mediated demethylation of H3K4me1 inhibited PP1α expression in oxycodone‐treated mice. (A–F) Dose–effect relationship between expressions of KDM5A, CoRest, H3K4me3/me2/me1, and dosage of oxycodone. (G–K) Time–effect relationship between expressions of CoRest, H3K4me3/me2/me1, and time at which oxycodone was administered. *p < 0.05, **p < 0.01, ***p < 0.001, vs. SAL+Veh group, one‐way ANOVA. LSD1 shRNA administration significantly increased H3K4me1 expression (L, M), lowered binding of LSD1 and CoRest (N, O) as well as binding of CoRest with the PP1α gene (P–R) in oxycodone‐treated mice. ***p < 0.001, vs. SAL+Veh group; ^### p < 0.001, vs. OXY + Veh group, one‐way ANOVA.