基于miRNA-34a-5p/PI3K/Akt信号通路探讨泄浊解毒方预防小鼠结直肠腺瘤的作用机制  

Mechanism of Xiezhuo Jiedu Recipe in Preventing Colorectal Adenoma in Mice Through miRNA-34a-5p/PI3K/Akt Signalling Pathway

作　　者：贾苏杰 孙超迪 张艺凡 郎晓猛[2,3] 刘建平 康欣[2,3] 任士杰 刘静远[1] JIA Sujie;SUN Chaodi;ZHANG Yifan;LANG Xiaomeng;LIU Jianping;KANG Xin;REN Shijie;LIU Jingyuan(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China;Key Laboratory of Integrated Chinese and Western Medicine for Gastroenterology Research(Hebei),Shijiazhuang 05001l,China)

机构地区：[1]河北中医药大学,石家庄050091 [2]河北省中医院,石家庄050011 [3]河北省中西医结合胃肠病研究重点实验室,石家庄050011

出　　处：《中国实验方剂学杂志》2024年第11期156-165,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医临床研究基地建设项目(国中医药办科技函[2018]131号);河北省自然科学基金和重点基础研究专项(H2019423077);政府资助临床医学优秀人才项目(ZF2023157);河北省中医药管理局科研计划项目(2022073);河北中医药大学研究生创新项目(XCXZZBS2023022)。

摘　　要：目的:通过生物信息学方法寻找并分析结直肠腺瘤(CRA)的关键微小RNA(miRNAs),筛选差异表达基因(DEGs)构建调控关系,推测泄浊解毒方预防CRA的作用机制,并通过动物实验进行验证。方法:从基因表达谱数据库(GEO)获取CRA患者肠黏膜组织miRNAs数据集GSE50194,通过GEO2R、Excel筛选差异表达的miRNAs;运用TargetScan、miRTarbase、miRDB数据库对差异表达的miRNAs进行靶基因预测并取得交集;通过STRING数据库及Cytoscape软件筛选关键DEGs,并利用TRRUST数据库预测下游结合转录因子;通过Metascape数据库对交集mRNA进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,通过DIANA TOOLS工具对关键miRNAs进行KEGG富集分析,选取关键信号通路进行动物实验验证。动物实验采用葡聚糖硫酸钠(DSS)饮用联合氧化偶氮甲烷(AOM)腹腔注射方法(10 mg·kg^(-1))诱导CRA小鼠模型建立,同时给予泄浊解毒方(9.62、19.24、38.48 g·kg^(-1))及阿司匹林(0.015 g·kg^(-1))灌胃干预9周。苏木素-伊红(HE)染色法观察结直肠组织病理变化;实时荧光定量聚合酶链式反应(Real-time PCR)检测腺瘤组织中miR-34a-5p mRNA表达量;蛋白免疫印迹法(Western blot)检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、磷酸化-PI3K(p-PI3K)、磷酸化-Akt(p-Akt)、B细胞淋巴瘤-2(Bcl-2)蛋白表达水平;免疫组织化学法(IHC)检测细胞周期蛋白D1(CCND1)的表达;原位末端标记法(TUNEL)检测腺瘤组织细胞凋亡情况。结果:GEO数据库筛选出GSE50194数据集,从CRA与正常肠黏膜组织中筛选出miR-34a-5p作为研究对象,筛选出93个DEGs,其GO和KEGG富集分析发现主要与转录调控复合体、RNA聚合酶Ⅱ转录调控复合物、酶联受体蛋白信号通路、DNA结合转录激活因子活性等生物学过程,癌症途径、PI3K/Akt信号通路等密切相关,miR-34a-5p主要汇集在PI3K/Akt信号通路、细胞周期、结直肠癌等途径。通过Matescape数据库筛选出5个�Objective: Key microRNAs(miRNAs) of colorectal adenoma(CRA) were identified and analyzed by bioinformatics methods, and differentially expressed genes(DEGs) were screened to construct regulatory relationships. The mechanism of Xiezhuo Jiedu recipe in preventing CRA was speculated and verified by animal experiments. Method: The miRNAs dataset GSE50194 was obtained from the Gene Expression Omnibus(GEO) database of intestinal mucosal tissue of CRA patients, and the differentially expressed miRNAs were screened by GEO2R and Excel. TargetScan, miRTarbase, and miRDB databases were used to predict the target genes of the differentially expressed miRNAs, and an intersection was obtained. Key DEGs were screened through the STRING database and Cytoscape software, and the TRRUST database was used to predict downstream binding transcription factors(TFs). The mRNA intersection was enriched by gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) in the Metascape database. DIANA TOOLS were applied to perform KEGG enrichment analysis of key miRNAs, and the key signaling pathways were selected for animal experiments. In animal experiments, the CRA mice model was established by using sodium glycan sulfate(DSS) drinking combined with intraperitoneal injection of azomethane oxide(AOM), and Xiezhuo Jiedu recipe and aspirin were given by intragastric administration at the same time. The experiment lasted for nine weeks. The pathological changes in intestinal tissue were observed by hematoxylin-eosin(HE) staining. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was used to detect the mRNA expression of miR-34a-5p in adenoma tissue. Protein expression levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), phosphoryl-PI3K(p-PI3K), phosphoryl-Akt(p-Akt), and B cell lymphoma(Bcl)-2 were detected by Western blot. The expression of Cyclin D1(CCND1) was detected by immunohistochemistry(IHC). In situ terminal transferase labeling(TUNEL) was used to detect apoptosis of adenoma tissue ce

关 键 词：生物信息学 结直肠腺瘤 泄浊解毒方 miR-34a-5p/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R287R22R2-031