Whole Transcriptome Sequencing of Peripheral Blood Shows That Immunity/GnRH/PI3K-Akt Pathways Are Associated With Opioid Use Disorder

Abstract

Background: Opioid use disorder (OUD), which is most commonly exhibited as addiction, is a persistent chronic disease that places a burden on families and society. Various peripheral traits have been linked to OUD in the past, but research on this topic is insufficient.

Methods: Seven male patients with OUD and 7 male healthy controls with matched demographic and clinical data were enrolled in this study. Peripheral blood RNA was used to construct an rRNA-removed library and a small RNA library. The peripheral transcriptomic differences between the two groups were investigated using RNA-seq. Differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) were identified by bioinformatics methods, and functional enrichment analysis with differentially expressed RNAs was performed to investigate the potential biological mechanisms of OUD.

Results: A total of 229 mRNAs (115 upregulated, 114 downregulated), 416 lncRNAs (191 upregulated, 225 downregulated), 17 circRNAs (16 upregulated, 1 downregulated) and 74 miRNAs (42 upregulated, 32 downregulated) were differentially expressed between the OUD group and the healthy control group. Functional enrichment analysis with differentially expressed mRNAs showed that immunity, GnRH secretion, and PI3K-Akt signaling pathways were associated with OUD. Immunity-, JAK-STAT-, and insulin-related pathways were enriched in functional enrichment analysis of target genes predicted by differentially expressed miRNAs.

Conclusion: We identified hundreds of differentially expressed genes that were enriched in immunity, GnRH secretion and PI3K-Akt signaling pathways. Some genes with significant changes might be used as potential biomarkers for progression and treatment of OUD.

Keywords: GnRH secretion; OUD; PI3K-Akt signaling pathway; RNA-seq; immunity; lncRNA.

Figure 1

Results of rRNA removed RNA-seq. (A) Principal component analysis results of all samples. The red dot in the figure represented the OUD group and blue dot represented the control group. (B) Volcano plot of all expressed genes. We set the threshold of differential expression as |log₂FoldChange| > 1.5 & p-value < 0.01. The blue dot, orange dot and gray dot represented the down-regulated genes (OUD/Control), up-regulated genes and non-significant changed genes, respectively. (C) Expression heatmap of all differentially expressed RNAs, including mRNA, lncRNAs and circRNAs. (D) GO biological process enrichment analysis results with differentially expressed mRNAs. (E) Protein-protein interaction results of differentially expressed genes. Gene names rendered in different colors indicated that they were in different protein interaction networks. (F) KEGG pathway enrichment analysis results with differentially expressed mRNAs.

Figure 2

Results of small RNA sequencing. (A) Principal component analysis results of all samples. The red dot in the figure represented the OUD group and blue dot represented the control group. (B) Volcano plot of all expressed miRNAs. We set the threshold of differential expression as |log₂FoldChange| > 1.5 & p-value < 0.01. The blue dot, orange dot and gray dot represented the down-regulated miRNAs (OUD/Control), up-regulated miRNAs and non-significant changed miRNAs, respectively. (C) Expression heatmap of all differentially expressed miRNAs. (D) KEGG enrichment result of targeted genes predicted by differentially expressed miRNAs.

Figure 3

Co-expression network of differentially expressed RNAs (mRNAs, lncRNAs, circRNAs and miRNAs) with differentially expressed RNAs in GnRH secretion (A) and PI3K-Akt signaling pathway (B). The green dots represented the differentially expressed mRNAs in GnRH secretion (A) and PI3K-Akt signaling pathway (B). The gray dots represented the differentially expressed ncRNAs (lncRNAs, circRNAs, miRNAs) coexpressed with differentially expressed mRNAs in these two pathways. (C) Differentially expressed miRNAs (orange box) and their targeted mRNAs (green dots).