农业生物技术学报2024，Vol.32Issue(3)：666-678,13.DOI:10.3969/j.issn.1674-7968.2024.03.016
miR-2779-x对MDCK细胞生长特性的影响及靶基因筛选验证
Effect of MiR-2779-x on the Growth Characteristics of MDCK Cells and Verification of Target Gene Screening
摘要
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.关键词
MDCK细胞/miR-2779-x/生长特性/靶基因Key words
MDCK cells/miR-2779-x/Growth characteristics/Target gene分类
农业科技引用本文复制引用
黄玲巍,杨迪,靳莉武,杨雅雯,王家敏,乔自林,阿依木古丽·阿不都热依木..miR-2779-x对MDCK细胞生长特性的影响及靶基因筛选验证[J].农业生物技术学报,2024,32(3):666-678,13.基金项目
甘肃省教育厅高校教师创新基金(2023B-058) （2023B-058）
中央高校基本科研业务费资金专项(31920230001) （31920230001）
