冠心病血清外泌体介导的lncRNA-miRNA-mRNA ceRNA调控网络研究及潜在靶向中药预测及实验验证  

Study of lncRNA-miRNA-mRNA ceRNA regulatory network mediated by serum exosomes in coronary heart disease and prediction and experimental validation of potential target herbal medicines

作　　者：马露 杨雷[3] 丁煌 李菀榆 谭维[1,2] 李艳玲 张燕燕 刘晓丹[1,2] 曾昭文 邓常清[1,2] 张伟 MA Lu;YANG Lei;DING Huang;LI Wan-yu;TAN Wei;LI Yan-ling;ZHANG Yan-yan;LIU Xiao-dan;ZENG Zhao-wen;DENG Chang-qing;ZHANG Wei(School of Integrative Medicine,Hunan University of Chinese Medicine;Key Laboratory of Hunan Province for Integrated Traditional Chinese and Western Medicine on Prevention and Treatment of Cardio-Cerebral Diseases;the First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院 [2]湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室 [3]湖南中医药大学第一附属医院,湖南长沙410208

出　　处：《中国药理学通报》2024年第6期1153-1164,共12页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82174218,81774032);国家自然科学基金区域创新发展联合基金重点项目(No U22A20377);湖南省科技创新领军人才项目(No 2023RC1066);湖南省科技创新计划资助(No 2023SK2021);湖南省研究生科研创新项目(No CX20220786,CX20220785);湖南中医药大学校级研究生创新课题(No 2023CX168)。

摘　　要：目的本研究采用生物信息学相关方法对冠心病(coronary heart disease,CHD)患者和正常人的血清外泌体测序数据分析,构建出竞争性内源性lncRNA-miRNA-mRNA(ceRNA)调控网络,并挖掘预测潜在的中药,并对ceRNA网络涉及的生物学过程和核心中药进行初步验证。方法利用exoRbase数据库获得差异基因的表达矩阵,结合生信方法构建ceRNA网络,并对网络中的差异的mRNA进行GO分析和KEGG分析,利用COREMINE数据库对ceRNA网络涉及的生物学过程及核心靶基因进行预测,筛选具有潜在治疗作用的中药;体外构建AVECs氧化损伤细胞模型,检测细胞骨架、成管功能、细胞增殖、LDH漏出率、ROS水平以及p-AKT、AKT、p-PI3K、PI3K蛋白表达情况,验证核心中药丹参治疗CHD的作用通路及靶点。结果与正常人比,CHD血清外泌体存在395个mRNA,80个miRNA,60个lncRNA差异基因,预测出80个miRNA,所构建ceRNA子网络,主要由21个lncRNA、80个miRNA、82个mRNA组成;AKT1、VEGFA、IL-1β等基因在网络中处于核心地位,异常表达的mRNA涉及细胞的氧化应激反应等生物学过程和PI3K/Akt等信号通路,丹参、川芎、三七等与CHD外泌体介导的生物学过程和核心基因最为密切;药物主要归属于补虚类、清热类和活血化瘀类,五味大多数归属于苦、甘和辛类,归经多集聚于心、肝、肾经;核心中药丹参的有效成分丹参酮IIA可以促进血管内皮细胞增殖、成管、骨架形成及修复,降低细胞LDH漏出率及ROS水平,促进p-AKT、p-PI3K蛋白的表达。结论CHD血清外泌体存在复杂的ceRNA调控网络转导,中药可通过多靶点干预治疗,丹参、川芎、三七等有望成为候选中药来源,其中丹参有效成分丹参酮IIA激活PI3K/Akt信号通路发挥对氧化应激损伤细胞的保护作用,治疗CHD。Aim To analyze serum exosome sequencing data from patients with coronary heart disease(CHD)and normal subjects by using bioinformatics-related methods to construct a competitive endogenous lncRNA-miRNA-mRNA(ceRNA)regulatory network,to mine the predicted potential Chinese medicines,and to perform preliminary validation of the biological processes and core Chinese medicines involved in the ceRNA network.Methods We used exoRbase database to obtain the expression matrix of differential genes,combined with the raw letter method to construct the ceRNA network,and performed GO analysis and KEGG analysis on the differential mRNAs in the network,and used COREMINE database to predict the biological processes and core target genes involved in the ceRNA network,and to screen the herbal medicines with potential therapeutic effects;AVECs oxidative damage cell model was constructed in vitro,and the cytoskeleton,tube-forming function,cell proliferation,LDH leakage rate,ROS level and p-AKT,AKT,p-PI3K and AKT protein expression were examined to verify the action pathways and targets of the core Chinese medicine Salvia miltiorrhiza for the treatment of coronary heart disease.Results Compared with normal subjects,395 mRNAs,80 miRNAs,60 lncRNA differential genes,and 80 miRNAs were predicted in serum exosomes of coronary heart disease,and the constructed ceRNA sub-network,mainly consisted of 21 lncRNAs,80 miRNAs,and 82 mRNAs;AKT1,VEGFA,IL1B and other genes in the network.The abnormally expressed mRNAs were involved in biological processes such as oxidative stress and signaling pathways such as PI3K/Akt,and Dan Shen,Chuanxiong and Panax notoginseng were most closely related to exosome-mediated biological processes and core genes in coronary heart disease.The active ingredients of tanshinone IIA,the core Chinese medicine,could promote vascular endothelial cell proliferation,tube formation,skeleton formation and repair,reduce LDH leakage rate and ROS level,and promote the expression of p-AKT and p-PI3K protein.Conclusion There is a compl

关 键 词：冠心病 血清外泌体 ceRNA lncRNA MIRNA MRNA 丹参酮IIA 

分 类 号：R284.1[医药卫生—中药学] R319[医药卫生—中医学] R329.24R342.22R541.4