应用RNA-Seq技术筛选不同浓度叶酸培养的QSG-7701细胞中的差异表达基因  

Primry study of differential mRNA expression patterns of QSG-7701 cells treated with different doses of folic acid by RNA-Seq

作　　者：张银玲[1] 薛赓[1] 孙树汉[1] 张毅[1] 

机构地区：[1]第二军医大学医学遗传学教研室,上海200433

出　　处：《东南国防医药》2016年第1期1-5,16,共6页Military Medical Journal of Southeast China

基　　金：国家自然科学基金资助项目(81272331)

摘　　要：目的在验证不同浓度叶酸培养影响人正常肝细胞系增殖、周期和迁移能力的基础上,应用转录组测序(RNASeq)技术筛选差异表达基因,为进一步探讨叶酸缺乏与基因差异表达以及正常肝细胞恶性转化之间的相关性打下基础。方法不同浓度叶酸培养(无叶酸组0 mg/L,正常叶酸组40 mg/L)的人正常肝细胞系QSG-7701 6个月,应用CCK-8实验检测细胞增殖、流式细胞技术检测细胞周期、细胞凋亡、Transwell小室检测细胞迁移;利用RNA-Seq技术对两组细胞中的mRNA进行检测,筛选差异mRNA;通过用实时定量PCR对RNA-Seq的结果进行验证,并结合统计学和生物信息学方法分析差异mRNA。结果无叶酸培养显著提高QSG-7701细胞的增殖能力,促进细胞由静止期进入分裂期,促进细胞的迁移能力;RNA-Seq检测得到含有16 774条差异mRNA的数据集,初步分析显示其中391条差异mRNA可能与不同浓度叶酸培养相关,其中上调的115条,下调的276条;随机对其中11条进行实时定量PCR验证,72.73%的结果与RNA-Seq一致;分析显示,差异mRNA相对应的基因与细胞周期等生理过程以及多种肿瘤的发生、发展密切相关。结论最终筛选出参与叶酸调控正常肝细胞恶性转化相关的基因,RNA-Seq技术能有效地用于差异基因地筛选。Objective To study the differential mRNA expression patterns of QSG-7701 cells that treated with folic acid which affects cell proliferation,cell cycle,cell apoptosis and transwell in order to investigate the relationship of folic acid deficiency,differential gene expression and malignant transformation of human normal hepatocytes by RNA-Seq. Methods Human normal hepatocytes QSG-7701 were treated with folic acid（ No folic acid group： 0 mg / L,Normal folic acid group： 40 mg / L） for 6 months. CCK-8,Flow cytometric and transwell were used to examine cell proliferation,cell cycle,cell apoptosis and cell migration. Real-time PCR was used to analyze and confirm RNA-Seq datas. The differential mRNA expression patterns was analyzed using bioinformatics and statistics.Results Folic acid deficiency promoted cell proliferation and cell migration. RNA-Seq results included 16 774 differential gene expression patterns and a total of 391 differential genes expressed in QSG-7701 cells were screened out,of which 115 were up regulated and276 were down regulated and 11 genes were random selected to confirm by Real-time PCR. As a result,a percent of 72. 73% genes were consisted with Real-time PCR. Analysis demonstrated that the abmormal gene had a close relationship with cell cycle,tumor inition and development. Conclusion Genes involved in normal cell malignant transformation can be effectively selected by RNA-Seq.

关 键 词：叶酸 QSG-7701细胞 转录组测序技术 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]