摘要
目的 构建Mindin低表达的Huh7细胞并对其进行转录组学研究,以探索Mindin的生物学作用.方法 设计并合成Mindin干扰序列,通过慢病毒载体GV248将其导入Huh7细胞,实时定量PCR检测对Mindin转录水平的抑制效果,获得Mindin低表达Huh7细胞并对该细胞及其对照细胞进行转录组学研究,生物信息学分析表达异常基因,Pathway分析Mindin可能参与的生物学功能.计量资料2组间比较采用t检验.结果 与对照细胞相比,含干扰序列的慢病毒感染Huh7细胞后,其Mindin mRNA的敲减效率为63.8%(P=0.003).Mindin稳定低表达的Huh7细胞迁移率明显高于对照细胞(3.511±0.538 vs 1.701±0.765,t=-3.355,P=0.03).与对照细胞相比,Mindin低表达Huh7细胞有2888个上调基因,2516个下调基因.取满足|log2 FC|>2.0且P<0.05的895个差异表达基因进行GO分析和Pathway分析.Pathway分析排名前10位的Mindin参与的生物学功能依次为:肿瘤信号通路、趋化因子信号通路、系统性红斑狼疮、甘油磷酸酯代谢、神经活性配体受体相互作用、肥厚性心肌病、甘油酯代谢、MAPK信号通路、凝血与补体级联反应和基底细胞癌.结论 成功构建了Mindin低表达Huh7细胞,Mindin的生物学功能涉及广泛,包括肿瘤、趋化因子和脂代谢等方面.
Objective To perform a transcriptomic study of Huh7 cells with low expression of Mindin,and to investigate the biological effects of Mindin.Methods The Mindin interference sequence was designed,synthesized,and introduced into Huh7 cells via the lentiviral vector GV248.Real-time quantitative PCR was used to measure the inhibitory effect on Mindin transcription level.Huh7 cells with low expression of Mindin were obtained and a transcriptomic study was performed for such cells and their control cells.Bioinformatics was used to analyze abnormally expressed genes,and pathway analysis was used to investigate the possible biological functions involving Mindin.The t -test was used for comparison of continuous data between groups.Results Compared with the control cells,Huh7 cells transfected with the lentivirus containing interference sequence had a knockdown efficiency of Mindin mRNA of 63.8%( P =0.003).Huh7 cells with stable low expression of Mindin had a significantly higher cell migration rate than the control cells (3.511±0.538 vs 1.701±0.765,t=-3.355,P =0.03).Compared with the control cells,Huh7 cells with low expression of Mindin had 2888 upregulated genes and 2516 downregulated genes.Gene ontology analysis and pathway analysis were performed for 895 differentially expressed genes with |log2FC|>2.0 and P <0.05.The pathway analysis showed that the top 10 biological functions involving Mindin were tumor signaling pathway,chemokine signaling pathway,systemic lupus erythematosus,glycerophospholipid metabolism,neuroactive ligand-receptor interaction,hypertrophic cardiomyopathy,glycerolipid metabolism,MAPK signaling pathway,complement and coagulation cascades,and basal cell carcinoma.Conclusion Huh7 cells with low expression of Mindin are successfully constructed.Mindin has wide biological functions including tumors,chemokines,and lipid metabolism.
作者
张欣
王沐淇
王文俊
李亚萍
石娟娟
李梅
贾晓黎
党双锁
ZHANG Xin;WANG Muqi;WANG Wenjun(Department of Infectious Diseases,The Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China)
出处
《临床肝胆病杂志》
CAS
北大核心
2019年第10期2272-2277,共6页
Journal of Clinical Hepatology
基金
国家自然科学基金(81500453)
陕西省社发科技攻关项目(S2015YFSF0363)
