基于蛋白质组学分析侧脑室微量注射GnIH对下丘脑蛋白的调节作用

Regulation of hypothalamic protein by microinjection of GnIH into lateral cerebral ventricle based on proteomics

目的观察侧脑室微量注射促性腺激素抑制激素(GnIH)对下丘脑蛋白质的调节作用。方法构建卵巢摘除术补充雌激素(OEP)大鼠模型(n=30),随机分为实验组和对照组各15只,侧脑室分别微量注射等量GnIH和生理盐水。给药6 h后取下丘脑,制备蛋白样品。应用液相色谱—串联质谱(LC-MS/MS)技术联合生物信息学分析工具[Omicsbean、基因本体论(GO)和京都蛋白与蛋白组百科全书(KEGG)]分析下丘脑蛋白质组分和差异蛋白质的分布、功能及作用机制。应用UALCAN数据库中CPTAC分析程序,分析关键蛋白在激素相关性肿瘤的表达情况。体外培养激素依赖性乳腺癌细胞系MCF-7,运用蛋白印迹检测关键蛋白的表达。结果鉴定出253个差异表达蛋白,其中129个蛋白上调,124个蛋白下调。GO结果显示,差异蛋白的生物过程主要在单体代谢过程和小分子代谢过程中富集,细胞成分主要分布在细胞质和细胞核,分子功能主要涉及小分子结合功能。KEGG结果显示,差异蛋白参与了代谢途径、糖酵解/糖异生、内分泌和其他因子调节的钙重吸收等10条重要信号通路。Omicsbean软件可视化蛋白—蛋白相互作用分析显示:关键蛋白GALM、ADPGK、PGM1、PFKL上调,ALDH2下调。CPTAC分析显示,关键蛋白GALM、ADPGK、PGM1、PFKL和ALDH2在乳腺癌中的表达高于正常乳腺组织。蛋白印迹结果显示,ADPGK在MCF-7表达下调。结论侧脑室微量注射GnIH后可引起OEP大鼠下丘脑蛋白的差异变化。

Objective To investigate the effect of microinjection of gonadotropin inhibitory hormone(GnIH)into lateral ventricle on hypothalamic protein.Methods Estrogen supplementation(OEP)for ovarian extraction rat models(n=30)were constructed,and were randomly divided into the experimental group and control group with 15 cases in each group.The rats were microinjected with the same amount of GnIH and saline into the lateral ventricle,respectively.After 6 h of administration,the hypothalami were removed to prepare the protein samples.Liquid chromatography tandem mass spectrometry(LC-MS/MS)combined with bioinformatics analysis tools[OmicsBean,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)]were used to analyze the hypothalamic protein components and distribution,function and mechanism of differential proteins.CPTAC analysis program in UALCAN database was used to analyze the expression of key proteins in hormone-associated tumor.The hormone-dependent breast cancer cell line MCF-7 was cultured in vitro,and the expression of key proteins was detected by Western blotting.Results A total of 253 differentially expressed proteins were identified,of which 129 proteins were up-regulated and 124 proteins were down-regulated.The results of GO showed that the biological process(BP)of differential proteins was mainly enriched in monomer metabolism and small molecule metabolism;cell component(CC)was enriched in the cytoplasm and intracytoplasm;molecular function(MF)was mainly involved in the binding of small molecules.The results of KEGG showed that differential proteins were involved in 10 important signaling pathways,including metabolic pathway,glycolysis/gluconeogenesis,and calcium reabsorption regulated by endocrine and other factors.Protein-protein interaction(PPI)analysis by omicsbean software showed that GALM,ADPGK,PGM1 and PFKL were up-regulated,ALDH2 was down-regulated.CPTAC analysis showed that the expression of key proteins GALM,ADPGK,PGM1,PFKL and ALDH2 in the cancer tissues were higher than those in the normal tissues.Western blotting experiment verified the down-regulation of ADPGK expression in estrogen receptor positive cell line MCF-7.Conclusion Microinjection of GnIH into the lateral ventricle can cause differentially changes in hypothalamic proteins of OEP rats.