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

作　　者：黄玲巍 杨迪[1,4] 靳莉武 杨雅雯 王家敏 乔自林[1,2,5] 阿依木古丽•阿不都热依木[1,2,3] HUANG Ling-Wei;YANG Di;JIN Li-Wu;YANG Ya-Wen;WANG Jia-Min;QIAO Zi-Lin;Ayimuguli ABUDUREYIMU(Biomedical Research Center/Engineering Research Center for Key Technologies and Industrialization of Cell-based Vaccines,Ministry of Education,Northwest Minzu University,Lanzhou 730030,China;Biomedical Research Center/Gansu Tech Innovation Center of Animal Cell,Northwest Minzu University,Lanzhou 730030,China;Life Science and Engineering College,Northwest Minzu University,Lanzhou 730030,China;Department of Experiment&Teaching,Northwest Minzu University,Lanzhou 730030,China;Biomedical Research Center/Key Laboratory of Bioengineering and Technology State Ethnic Affairs Commission,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]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 [2]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 [3]西北民族大学生命科学与工程学院,兰州730030 [4]西北民族大学实验教学部,兰州730030 [5]西北民族大学生物医学研究中心/生物工程与技术国家民委重点实验室,兰州730030

出　　处：《农业生物技术学报》2024年第3期666-678,共13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(31920230001)。

摘　　要：犬肾细胞系MDCK(Madin Darby Canine Kidney)是生产流感病毒疫苗最常用的细胞系之一,miR-2779-x作为非编码RNA能够抑制MDCK细胞的自发肿瘤转化。本研究基于慢病毒系统构建过表达及敲低miR-2779-x的稳转细胞株,利用细胞增殖检测试剂盒CCK-8(Cell Counting Kit-8)、细胞凋亡和细胞周期检测试剂盒以及细胞划痕试验、软琼脂克隆试验对稳转细胞株进行检测。结果显示,miR-2779-x过表达使细胞增殖能力和相对迁移率显著降低(P<0.01)、侵袭能力增加(P<0.01),敲低细胞株的结果与之相反;过表达细胞株的早期凋亡率显著提高(P<0.01),敲低细胞株的晚期凋亡率显著提高(P<0.01);miR-2779-x过表达使细胞在S期发生阻滞,敲低细胞株在G0/G1期发生阻滞。半数组织培养感染剂量(50%tissue culture infective dose,TCID50)检测结果显示,转入过表达及敲低miR-2779-x慢病毒的MDCK细胞对甲型H1N1流感病毒株的敏感性无显著变化。通过qRT-PCR和Western blot验证miR-2779-x靶基因的表达变化,发现多个与转化致瘤和生长相关的基因受到miR-2779-x负调控,推测靶基因CASP9(caspase 9)可能作为凋亡通路因子与PI3K/AKT信号通路共同调控MDCK细胞增殖。本研究为建立新型MDCK细胞系提供了新思路,可为流感疫苗生产提供更好的细胞基质。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 miR2779-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 

分 类 号：S852.23[农业科学—基础兽医学]