Screening and Validation of Tumorigenicity-related CircRNAs in MDCK Cells
YANG Di1,2,3,4, SHI Jia-Chen2,3, HUANG Ling-Wei2,3, WANG Jia-Min2,3, MA Yu-Mei5, QIAO Zi-Lin2,3, CUI Yan1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Engineering Research Center for Key Technologies and Industrialization of Cell-based Vaccines, Ministry of Education, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China; 3 Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China; 4 Department of Experiment & Teaching, Northwest Minzu University, Lanzhou 730030, China; 5 Gansu Provincial Bioengineering Materials Engineering Research Center, Lanzhou Minhai Bio-engineering Co., Ltd., Lanzhou 730010, China
Abstract 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 RNA-seq 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 non-tumorigenic MDCK cells were extracted using TRIzol method, and the expression levels of 6 differential circRNAs were verified by qPCR. The results showed that 3 491 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.
YANG Di,SHI Jia-Chen,HUANG Ling-Wei, et al. Screening and Validation of Tumorigenicity-related CircRNAs in MDCK Cells[J]. 农业生物技术学报, 2024, 32(9): 2088-2099.
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