摘要
目的检测甲基苯丙胺依赖大鼠和健康大鼠血清外泌体中微小RNA(miR)-181a-5p的表达,并对miR-181a-5p进行靶基因预测及相关生物信息学分析。方法用甲基苯丙胺复制大鼠条件性位置偏爱(CPP)模型。按照体重将雄性SD大鼠随机分为2组(每组10只):正常组(0.9%NaCl),实验组(2mg·kg^(-1)甲基苯丙胺)。实验组上午8:00皮下注射甲基苯丙胺,下午16:00皮下注射0.9%NaCl溶液,连续4 d。正常组同时皮下注射等体积0.9%NaCl溶液。实验组与正常组上午处置后,立即置于白箱训练1 h;下午皮下注射0.9%NaCl溶液后,立即置于黑箱训练1 h,连续训练4 d。在末次处置1 d后,进行位置偏爱测定,记录大鼠15 min内在白箱中停留时间及运动路程。用RT-qPCR技术检测大鼠血清外泌体中miR-181a-5p的表达水平;以Target Scan、miRanda及miRBD预测miR-181a-5p靶基因;用DAVID数据库对其靶基因进行功能聚类分析(GO)和生物通路(KEGG pathways)分析;以STRING在线分析软件对其靶基因蛋白进行相互作用分析。结果 CPP训练后,2组在白箱中停留时间(s)及运动路程(cm):正常组分别为202.67±28.24,3349.26±412.28;实验组分别为460.47±66.71,4475.97±585.74。实验组与正常组比较,差异均有统计学意义(均P<0.01)。正常组、实验组血清外泌体中miR-181a-5p表达分别为3.39±1.79,10.23±4.32,组间差异有统计学意义(P<0.01)。预测得到miR-181a-5p靶基因108个,其功能主要富集于神经元的生成和分化、基因表达调控及谷氨酸转运等生物学过程,涉及内质网蛋白加工信号通路。miR-181a-5p靶基因编码蛋白之间存在复杂的相互作用,其中RAD23B、PRKCD、MAP2K1、NCBP1、GABRA1等在蛋白互作网络中具有核心地位。结论miR-181a-5p可能通过调控基因表达及谷氨酸转运等生物过程,作用于内质网蛋白加工信号通路,进而调节下游靶蛋白参与甲基苯丙胺成瘾的发生过程。
Objective To detect the expression of microRNA（miR）-181a-5p in serum exosomes of methamphetamine-dependent rats and healthy rats,and to predict target gene of miR-181a-5p and related bioinformatics analysis. Methods The conditioned place preference（ CPP） model was established by methamphetamine： male SD rats were randomly divided into two groups（ ten rats in each group） ： normal group（ 0. 9% NaCl）, and experimental group（ methamphetamine 2 mg·kg^（-1））. The experimental group was subcutaneously injected methamphetamine at 8 am,injected saline at 4 pm for four days. The normal group was injected subcutaneously with equal volume of normal saline. In the morning,after the experimental group（ normal group） were injected subcutaneously with methamphetamine（ saline）,they were immediately put in the white side for one hour. In the afternoon,after rats were injected subcutaneously with saline,they were immediately put in the black side for one hour. This training was carried out continuously for 4 days. After 1 day of the last subcutaneous injection of methamphetamine（ saline）,the conditioned place preference was measured. The residence time and movement distance in the white side were recorded within 15 min. The expression of miR-181a-5p in rats＇ serum exosomes was detected by RT-qPCR technique. The miR-181a-5p target gene was predicted by Target Scan,miRanda and miRBD. The target gene was analyzed by Gene Ontology（ GO） analysis and Kyoto Encyclopedia of Genomes and Genes pathways（ KEGG pathways）with DAVID Data Base. The target gene protein was carried out interactive analysis by STRING online analysis software.Results After CPP training,the residence time（ s） and the moving distance（ cm） of normal group in the white side were 202. 67 ± 28. 24 and 3349. 26 ± 412. 28 respectively; the experimental group were 460. 47 ± 66. 71 and 4475. 97 ± 585. 74 respectively. Compared with the normal group,the residence time and the moving distance of the experimental group were significantly increased in the white side（ P 0. 01）. The expression of miR-181a-5p in serum exosomes in the normal group and the experimental group was 3. 39 ± 1. 79,10. 23 ± 4. 32 respectively.Compared with the normal group,the expression of miR-181a-5p in the experimental group had increased significantly（ P 0. 01）. The 108 miR-181a-5p target genes were predicted to be rich in biological processes such as neuronal generation and differentiation,gene expression regulation and glutamate transport,and involved in the pathogenesis of protein processing in endoplasmic reticulum. MiR-181 a-5 p target gene encodes a complex interaction between proteins,including RAD23B,ATXN3,PRKCD,MAP2K1,NCBP1,GABRA1 and so on. They played a core position in the protein-protein interaction（ PPI） network. Conclusion miR-181a-5p may regulate the downstream target protein and participate in the development of methamphetamine addiction by regulating gene expression,glutamate transport and participate in the pathway of protein processing in endoplasmic reticulum.
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2018年第1期45-48,共4页
The Chinese Journal of Clinical Pharmacology
基金
国家自然科学基金资助项目(81673628
81229003
81403063)
广东省科技计划基金资助项目(201707010287)
广东省自然科学基金资助项目(2014A030310251)
