MDCK细胞成瘤性相关环状RNA的筛选与验证  

Screening and Validation of Tumorigenicity-related CircRNAs in MDCK Cells

作　　者：杨迪 石嘉琛[2,3] 黄玲巍 王家敏 马玉梅[5] 乔自林 崔燕[1] YANG Di;SHI Jia-Chen;HUANG Ling-Wei;WANG Jia-Min;MA Yu-Mei;QIAO Zi-Lin;CUI Yan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Engineering Research Center for Key Technologies and Industrialization of Cell-based Vaccines,Ministry of Education,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Gansu Tech Innovation Center of Animal Cell,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Department of Experiment&Teaching,Northwest Minzu University,Lanzhou 730030,China;Gansu Provincial Bioengineering Materials Engineering Research Center,Lanzhou Minhai Bio-engineering Co.,Ltd.,Lanzhou 730010,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]西北民族大学生物医学研究中心细胞基质疫苗关键技术与产业化教育部工程研究中心,兰州730030 [3]西北民族大学生物医学研究中心甘肃省动物细胞技术创新中心,兰州730030 [4]西北民族大学实验教学部,兰州730030 [5]兰州民海生物工程有限公司甘肃省生物工程材料工程研究中心,兰州730010

出　　处：《农业生物技术学报》2024年第9期2088-2099,共12页Journal of Agricultural Biotechnology

基　　金：甘肃省教育厅高校教师创新基金(2023B-058);中央高校基本科研业务费资金专项(31920240063);甘肃省科技计划(23YFFA0071)。

摘　　要：在应用犬(Canis lupus familiaris)肾细胞系MDCK (Madin-Darby canine kidney)制备禽流感等兽用疫苗中发现,MDCK细胞系存在成瘤性。本研究以高成瘤性和无成瘤性的MDCK细胞为研究对象,通过RNA-seq高通量测序技术筛选获得差异表达的环状RNA (circular RNA, circRNA),并对差异表达circRNA的来源基因进行GO和KEGG分析;进一步使用数据库StarBase和mirTarBase分别预测成瘤性相关差异circRNA的靶标微小RNA (microRNA, miRNA)和mRNA,使用Cytoscape软件绘制circRNAmiRNA-mRNA共表达网络。利用TRIzol法提取高成瘤性和无成瘤性的MDCK细胞总RNA,利用q PCR验证6个差异circRNA的表达水平。结果显示,从高成瘤性和无成瘤性的MDCK细胞中鉴定出3 491个circRNA,长度为200~800 nt;共筛选出27个差异表达的circRNA,其中18个上调表达、9个下调表达。GO、KEGG和Reactome富集分析表明,差异表达的circRNA与ATP和氨基酸代谢相关酶的活性及结合等相关,主要富集的信号通路为黏附连接、细胞迁移以及多种代谢相关信号通路;内源竞争RNA(competing endogenous RNA, ceRNA)网络构建得到4组MDCK细胞成瘤性相关的关键circRNA-miRNAmRNA调控网络,分别为novel_circ_002222/novel-m0145-3p/动力蛋白激活蛋白1 (dynactin 1, Dctn1)、novel_circ_002222/miR-197-x/Dctn1、novel_circ_001205/novel-m0580-5p/膜联蛋白A11 (annexin A11,ANXA11)和novel_circ_003257/novel-m0675-3p/RNA结合蛋白48 (RNA-binding protein 48, RBM48)。上述结果表明,MDCK细胞成瘤性相关的差异表达circRNA主要富集在跨膜物质运输和能量代谢相关通路,可能在MDCK细胞能量摄入和代谢过程中发挥作用。本研究为揭示circRNA参与MDCK细胞成瘤的机制、建立基因工程无成瘤性MDCK细胞系提供研究基础和技术支持。In the preparation of animal vaccines such as avian influenza using the canine(Canis lupus familiaris)kidney cell line Madin-Darby canine kidney(MDCK),it was found that the MDCK cell line has tumorigenicity.In this study,highly tumorigenic and non-tumorigenic MDCK cells were selected for RNAseq high-throughput sequencing.The differentially expressed circular RNA(circRNA)were obtained,and their source genes were analyzed by GO and KEGG.The StarBase and mirTarBase databases were used to predict target microRNA(miRNA)and mRNA of tumorigenic circRNA.Total RNA of tumorigenic and nontumorigenic MDCK cells were extracted using TRIzol method,and the expression levels of 6 differential circRNAs were verified by qPCR.The results showed that 3491 circRNAs were identified with the length of 200~800 nt.Among the screened out 27 differentially expressed circRNAs,18 were up-regulated and 9 were down-regulated.GO,KEGG and Reactome enrichment analysis revealed that differentially expressed circRNAs were associated with ATP activity and amino acid metabolism-related enzymes,linked to adhesion,migration,and various metabolic pathways.The network construction of competing endogenous RNA(ceRNA)illustrated 4 key circRNA-miRNA-mRNA regulatory network related to MDCK cell tumorigenicity,novel_circ_002222/novel-m0145-3p/dynactin 1(Dctn1),novel_circ_002222/miR-197-x1/Dctn1,novel_circ_001205/novel-m0580-5p/annexin A11(ANXA11),and novel_circ_003257/novel-m0675-3p/RNA-binding protein 48(RBM48).The above results indicate that differentially expressed circRNAs related to tumorigenicity in MDCK cells are mainly enriched in transmembrane material transport and energy metabolism-related pathways,which might play a role in energy uptake and metabolism in MDCK cells.This study provides a research foundation and technical support for revealing the mechanism of circRNA involvement in MDCK cell tumorigenesis and establishing genetically engineered non-tumorigenic MDCK cell lines.

关 键 词：MDCK(Madin-Darby canine kidney) 环状RNA(circRNA) 成瘤性 

分 类 号：S852.23[农业科学—基础兽医学]