Identification and characterization of N6-methyladenosine circular RNAs in the spinal cord of morphine-tolerant rats

Abstract

Morphine tolerance (MT) is a tricky problem, the mechanism of it is currently unknown. Circular RNAs (circRNAs) serve significant functions in the biological processes (BPs) of the central nervous system. N6-methyladenosine (m⁶A), as a key post-transcriptional modification of RNA, can regulate the metabolism and functions of circRNAs. Here we explore the patterns of m⁶A-methylation of circRNAs in the spinal cord of morphine-tolerant rats. In brief, we constructed a morphine-tolerant rat model, performed m⁶A epitranscriptomic microarray using RNA samples collected from the spinal cords of morphine-tolerant rats and normal saline rats, and implemented the bioinformatics analysis. In the spinal cord of morphine-tolerant rats, 120 circRNAs with different m⁶A modifications were identified, 54 of which were hypermethylated and 66 of which were hypomethylated. Functional analysis of these m⁶A circRNAs found some important pathways involved in the pathogenesis of MT, such as the calcium signaling pathway. In the m⁶A circRNA-miRNA networks, several critical miRNAs that participated in the occurrence and development of MT were discovered to bind to these m⁶A circRNAs, such as miR-873a-5p, miR-103-1-5p, miR-107-5p. M⁶A modification of circRNAs may be involved in the pathogenesis of MT. These findings may lead to new insights into the epigenetic etiology and pathology of MT.

Keywords: N6-methyladenosine; bioinformatics analysis; circRNAs; microarray; morphine tolerance.

FIGURE 1

The animal model and the overall features of m⁶A-circRNA in morphine-tolerant rats. (A) Morphine-induced antinociception was assessed through the tail-flick test, and tail-flick latency was converted to %MPE (n = 4), ***P < 0.001 compared with the NS group using Two-way repeated measures of ANOVA followed by Bonferroni’s post-hoc test. (B) Expression of RNA demethylase and methyltransferase determined by qRT-PCR. (n = 4) **P < 0.01, ***P < 0.001 compared with the NS group using a two-tailed unpaired Student’s t-test. (C) Expression of RNA methylase determined by qRT-PCR (n = 4). *P < 0.05 compared with the NS group using a two-tailed unpaired Student’s t-test. (D) Hierarchical clustering shows global m⁶A-methylated circRNAs between the MT group and NS group (n = 4).

FIGURE 2

Distribution of differentially methylated circRNAs. (A) The volcano plot shows the distribution of differentially methylated circRNAs (Fold change ≥ 1.5 and P < 0.05) between the MT group and the NS group. (B) The chromosome’s origins for host genes of differentially m⁶A-methylated circRNAs. (C) The genomic origins of differentially m⁶A-methylated circRNAs. (D) The length distribution of differentially m⁶A-methylated circRNAs. (E) The enriched pathways of the host gene of the differentially m⁶A-methylated circRNAs through KEGG analyses. (F) The top 15 enriched GO terms of the host gene of the hypermethylated circRNAs. GO terms include biological process (BP) analysis, cellular component (CC) analysis, and molecular function (MF) analysis. (G) The top 15 enriched GO terms of the host gene of the hypomethylated circRNAs.

FIGURE 3

Joint analysis of m⁶A methylation and expression patterns of circRNAs in morphine tolerance. (A) The nine-quadrant graph shows the relationship between m⁶A quantity and the expression of circRNAs. (B) Cumulative distribution of circRNAs expression between MT group and NS group for m⁶A-circRNAs (blue) and non-m⁶A- circRNAs (red). (C) The upset diagram shows the m⁶A quantity, expression, and m⁶A level of circRNAs and presents 14 different methylation and expression patterns of circRNAs. (D) The dot plot shows the distribution of 96 circRNA with both dysregulated m⁶A quantity and m⁶A level. The orange dots (hyper-up) show 27 circRNAs were hypermethylated and up-expressed, 2 navy blue dots (hypo-down) represent circRNAs were hypomethylated and down-expressed; 1 black dot (hypo-up) represent circRNA were hypomethylated and up-expressed; the light blue dots (hypo-no) represent 66 circRNAs with decreased m6A modification quantity but unchanged expression levels.

FIGURE 4

Construction of circRNA–miRNA networks in morphine tolerance using circRNAs with differences in both m⁶A quantity and m⁶A modification level.

FIGURE 5

M⁶A levels of the four circRNAs involved in the circRNA–miRNA networks were verified by MeRIP-qPCR. Relative m⁶A methylation level was calculated as the percentage of modified transcripts in all transcripts. P-values were calculated using Student’s t-test (n = 4). **P < 0.01, ***P < 0.001 compared with the NS group using a two-tailed unpaired Student’s t-test.