苍耳亭与肝癌细胞MHCC97H共培养后细胞外泌体测序及功能分析  

Sequencing Screening and Functional Analysis of Exosomes in Xanthatin Treated MHCC97H Hepatoma Cancer Cells

作　　者：吴育[1,2] 汪洁 魏晨旭[2] 张硕 李伟东[2] WU Yu;WANG Jie;WEI Chenxu;ZHANG Shuo;LI Weidong(Department of Pharmacy,Nantong Hospital Affiliated to Nanjing University of Chinese Medicine,Nantong 226001,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Taizhou Campus,Nanjing University of Chinese Medicine,Taizhou 225300,China)

机构地区：[1]南京中医药大学南通附属医院药剂科,南通226000 [2]南京中医药大学药学院,南京210023 [3]南京中医药大学泰州分校,泰州225300

出　　处：《医药导报》2023年第6期807-813,共7页Herald of Medicine

基　　金：南通市科技局资助项目(JCZ20166);南通市卫健委资助项目(MA2020005);常州市四药资助项目(NTYX2016)。

摘　　要：目的筛选苍耳亭与MHCC97H共培养过程中差异miRNAs并进行验证,为寻找肝癌治疗靶点提供线索。方法将苍耳亭与肝癌细胞MHCC97H共培养24 h,利用差速超离心法分离出外泌体。通过透射电镜观察外泌体的典型结构以及Western blotting分析外泌体标志性蛋白CD9和HSP70的表达来验证分离的是外泌体;检测苍耳亭与MHCC97H共培养后细胞外泌体的miRNA谱。筛选苍耳亭与MHCC97H细胞共培养和未共培养的MHCC97H细胞外泌体的差异miRNAs,通过GO和KEGG分析表征差异miRNAs的功能,分析差异miRNAs的潜在抗肿瘤意义。利用实时荧光定量PCR(qRT-PCR)对筛选的4种差异miRNAs进行验证。结果共鉴定出78个差异miRNAs,其中36个上调miRNA,42个下调miRNA。差异基因主要与肿瘤代谢通路相关。qRT-PCR实验结果显示,细胞外泌体let-7f-5p、let-7b-5p、miR-192-5p和miR-197-3p表达上调,与miRNA测序结果一致。结论苍耳亭可调节肿瘤细胞来源的外泌体miRNA,这些外泌体对抑制肝癌具有重要意义。Objective To screen and verify differential miRNAs during the process of co-culturing MHCC97H hepatoma cells with xanthatin,which provided clues for the search for therapeutic targets for liver cancer.Methods MHCC97H cells were co-cultured with xanthatin for 24 h and then the EVs were isolated from conditioned medium by differential ultra-centrifugation.The typical structures of exosomes were observed by transmission electron microscopy,and the expression of exosomes marker proteins CD9 and HSP70 was analyzed by Western blotting.MicroRNA(miRNA)sequencing was used to establish the miRNA profiles of xanthatin-treated MHCC97H.Differential miRNAs were screened between xanthatin treated MHCC97H cells and untreated MHCC97H cells.Target genes of the differential miRNAs were predicted by MiRanda software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were further performed to characterize the function of differential miRNAs.In addition,critical miRNAs identified by small RNA sequencing were validated by quantitative real-time PCR(qRT-PCR).Results A total of 78 differential miRNAs were identified,including 36 upregulated and 42 downregulated miRNAs.GO and KEGG analyses showed that the genes were related to pathways in cancer and metabolism,such as apoptosis,inflammation,metabolism,endocrine process,TGF-βpathway,and Wnt signaling pathway.qRT-PCR demonstrated that the expression of let-7f-5p,let-7b-5p,miR-192-5p and miR-197-3p were upregulated in xanthatin-treated MHCC97H cells,which was in consistent with miRNA sequencing.Conclusion The xanthatin could regulate tumor-derived exosomal miRNAs.These exosomes play an important role in inhibiting hepatocellular carcinoma.

关 键 词：苍耳亭 肝癌 miRNA测序 

分 类 号：R285[医药卫生—中药学]