Table1_Identification of miRNA-mediated gene regulatory networks in L-methionine exposure counteracts cocaine-conditioned place preference in mice.DOCX

Background and Aims: Methionine has been proven to inhibit addictive behaviors of cocaine dependence. This study aimed to identify the potential mechanisms of MET relating to its inhibitory effects on cocaine induced cellular and behavioral changes.
Methods: MRNA and miRNA high-throughput sequencing of the prefrontal cortex in a mouse model of cocaine conditioned place preference (CPP) combined with L-methionine was performed. Differentially expressed miRNAs (DE-miRNAs) and differentially expressed genes (DEGs) regulated by cocaine and inhibited by L-methionine were identified. DEGs were mapped to STRING database to construct a protein-protein interaction (PPI) network. Then, the identified DEGs were subjected to the DAVID webserver for functional annotation. Finally, miRNA-mRNA regulatory network and miRNA-mRNA-TF regulatory networks were established to screen key DE-miRNAs and coregulation network in Cytoscape.
Results: Sequencing data analysis showed that L-methionine reversely regulated genes and miRNAs affected by cocaine. Pathways associated with drug addiction only enriched in CS-down with MC-up genes targeted by DE-miRNAs including GABAergic synapse, Glutamatergic synapse, Circadian entrainment, Axon guidance and Calcium signaling pathway. Drug addiction associated network was formed of 22 DEGs including calcium channel (Cacna1c, Cacna1e, Cacna1g and Cacng8), ephrin receptor genes (Ephb6 and Epha8) and ryanodine receptor genes (Ryr1 and Ryr2). Calcium channel gene network were identified as a core gene network modulated by L-methionine in response to cocaine dependence. Moreover, it was predicted that Grin1 and Fosb presented in TF-miRNA-mRNA coregulation network with a high degree of interaction as hub genes and interacted calcium channels.
Conclusion: These identified key genes, miRNA and coregulation network demonstrated the efficacy of L-methionine in counteracting the effects of cocaine CPP. To a certain degree, it may provide some hints to better understand the underlying mechanism on L-methionine in response to cocaine abuse.

研究背景与目的：已有研究证实，甲硫氨酸（Methionine）可抑制可卡因依赖（cocaine dependence）相关的成瘾行为。本研究旨在阐明L-甲硫氨酸（L-methionine）发挥可卡因诱导的细胞与行为改变抑制作用的潜在分子机制。
研究方法：本研究针对可卡因条件性位置偏好（Cocaine Conditioned Place Preference, CPP）小鼠模型，联合给予L-甲硫氨酸，对其前额叶皮层进行信使RNA（messenger RNA, mRNA）与微小RNA（microRNA, miRNA）高通量测序。筛选受可卡因调控且可被L-甲硫氨酸抑制的差异表达微小RNA（differentially expressed miRNAs, DE-miRNAs）与差异表达基因（differentially expressed genes, DEGs）。将差异表达基因映射至STRING数据库以构建蛋白质相互作用（protein-protein interaction, PPI）网络；随后通过DAVID在线服务器对差异表达基因进行功能注释；最终利用Cytoscape软件构建微小RNA-信使RNA调控网络与微小RNA-信使RNA-转录因子（transcription factor, TF）调控网络，以筛选关键差异表达微小RNA及共调控网络。
研究结果：测序数据分析显示，L-甲硫氨酸可反向调控可卡因影响的基因与微小RNA。仅在受差异表达微小RNA靶向的、可卡因给药组下调且L-甲硫氨酸给药组上调的基因中，富集到与药物成瘾相关的通路，包括γ-氨基丁酸能突触（GABAergic synapse）、谷氨酸能突触（Glutamatergic synapse）、昼夜节律调节（Circadian entrainment）、轴突导向（Axon guidance）以及钙信号通路（Calcium signaling pathway）。成瘾相关调控网络由22个差异表达基因构成，涵盖钙通道基因（Cacna1c、Cacna1e、Cacna1g及Cacng8）、Eph家族受体基因（Ephb6与Epha8）以及雷诺丁受体基因（Ryr1与Ryr2）。钙通道基因网络被鉴定为L-甲硫氨酸调控可卡因依赖应答的核心基因网络。此外，预测显示谷氨酰受体1（Grin1）与FBJ鼠骨肉瘤病毒癌基因同源物B（Fosb）作为枢纽基因，存在于高互作度的转录因子-微小RNA-信使RNA共调控网络中，并可与钙通道基因发生相互作用。
研究结论：本研究鉴定的关键基因、微小RNA及共调控网络，证实了L-甲硫氨酸可有效拮抗可卡因条件性位置偏好诱导的效应。该研究在一定程度上可为阐明L-甲硫氨酸应答可卡因滥用的潜在分子机制提供新思路。