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| 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 |
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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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Received: 28 July 2023
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Corresponding Authors:
** Ayimgul80@xbmu.edu.cn
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| About author:: * These authors contributed equally to this work |
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