Effect of MiR-2779-x on the Growth Characteristics of MDCK Cells and Verification of Target Gene Screening
HUANG Ling-Wei1,2,3,*, YANG Di1,4,*, JIN Li-Wu1,2,3, YANG Ya-Wen1,3,5, WANG Jia-Min1,2,5, QIAO Zi-Lin1,2,5, Ayimuguli ABUDUREYIMU1,2,3,**
1 Biomedical Research Center/Engineering Research Center for Key Technologies and Industrialization of Cell-based Vaccines, Ministry of Education, Northwest Minzu University, Lanzhou 730030, China; 2 Biomedical Research Center/Gansu Tech Innovation Center of Animal Cell, Northwest Minzu University, Lanzhou 730030, China; 3 Life Science and Engineering College, Northwest Minzu University, Lanzhou 730030, China; 4 Department of Experiment & Teaching, Northwest Minzu University, Lanzhou 730030, China; 5 Biomedical Research Center/Key Laboratory of Bioengineering and Technology State Ethnic Affairs Commission, Northwest Minzu University, Lanzhou 730030, China
Abstract Madin Darby Canine Kidney (MDCK) cell line is one of the most commonly used cell lines to produce Influenza virus vaccine, and miR-2779-x, as a non-coding RNA, can inhibit spontaneous tumor transformation in MDCK cells. In this study, stable cell lines with overexpression and knockdown of miR-2779-x were constructed based on the lentivirus system, and the transfected cells were detected by cell proliferation detection kit CCK-8 (Cell Counting Kit-8), cell apoptosis and cell cycle detection kit, as well as cell scratch test and soft agar cloning test. The results showed that overexpression of miR-2779-x significantly decreased cell proliferation and relative mobility (P<0.01), and increased cell invasion (P<0.01), while knockdown cell lines showed opposite results. The early apoptosis rate of overexpressed cell lines and the late apoptosis rate of knockdown cell lines were significantly increased (P<0.01). miR-2779-x overexpression caused cell block in S phase, and knockdown cell lines were blocked in G0/G1 phase. Measured at 50% tissue culture infective dose (TCID50), the sensitivity of MDCK cells with miR-2779-x lentivirus overexpression and knockdown did not change significantly to Influenza virus A H1N1. The expression changes of miR-2779-x target genes were verified through qRT-PCR and Western blot, and multiple genes related to transformation, tumorigenesis, and growth were found to be negatively regulated by miR-2779-x. It is speculated that the target gene CASP9 (caspase 9) might act as an apoptotic pathway factor and co-regulate MDCK cell proliferation with PI3K/AKT signaling pathway. This study provides a new approach for establishing a novel MDCK cell line, which can provide a better cell matrix for influenza vaccine production.
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