摘要
目的:探讨ebv-miRNA在低分化NPC细胞株C666-1(EBV+)和CNE-2Z(EBV表达水平随传代次数增加而逐渐降低)中的差异性表达进而分析其功能。方法:常规培养NPC细胞株C666-1和CNE-2Z,分别提取总RNA并进行质检;RT-PCR检测LMP-1和LMP-2A mRNA的表达以判断EBV的感染情况;miRNA芯片技术检测EBV miRNAs的表达并对其表达谱进行差异性分析;荧光定量RT-PCR进行验证;复习文献了解ebv-miRNA调控的靶基因以了解其功能。结果:从C666-1和CNE-2Z两株细胞抽提的总RNA纯度高,A260/A280>2.0,A260/A230>2.1,RNA完整性好。C666-1具有比CNE-2Z更高的LMP-1和LMP-2A mRNA表达。ebv-miRNA表达谱的差异分析表明39个ebv-miRNAs中19个在两株细胞显著差异性表达;荧光定量RT-PCR结果证实ebv-miR-BHRF1-1和ebv-miR-BART14*在C666-1中表达升高而ebv-miR-BART8*表达降低,与芯片结果趋势一致;复习文献,EBV miRNAs功能远不清楚,少数已知的靶基因涉及信号转导、转录调控、细胞凋亡、增殖、免疫反应和病毒DNA复制等方面。结论:ebv-miRNA在低分化NPC中具有差异性表达并广泛参与基因调控。不同NPC细胞中EBV miRNA表达差异预示着基于ebv-miRNA的NPC个性化治疗的可能性。
Objective ： Aberrant expression of microRNAs （miRNAs） is involved in the progression of carcinomas. Epstein Barr virus （EBV） encodes near 40 miRNAs. This study was aimed to investigate the differential expression of EBV -encoded miRNAs in two poor- differentiated nasopharyngeal carcinomas （NPC） cell line C666 -1 and CNE - 2Z, and to analyze targets of the ebv - miRNAs. Methods： Human NPC cell line C666 - 1 （ EBV＋ ） and CNE - 2Z （EBV may lose due to the mass passages） were cultured, total RNA was isolated and examined, mRNAs of LMP -1 and LMP- 2A were assessed by RT- PCR to identify the infection of EBV, and the expression profiles of ebv- miRNAs was detected with miRNA microarray chip and compared array results was confirmed by real - time PCR. Ebv - miRNA targets were analyzed by literature review. Results ： The purification of total RNA from C666 - 1 and CNE-2Z were high,and the expression of mRNAs of LMP- 1 and LMP-2A was detected in C666-1 and CNE - 2Z. According to microarray screening, 19/39 ebv -miRNAs were differentially expressed. Real -time PCR revealed that ebv - miR - BHRF1 - 1 and ebv - miR - BART14 ＊ were up - regulated in C666 - 1, while ebv - miR - BART8 ＊ was down - regulated, these results were coincidence with the tendency of microarray. Up to date, limitable ebv - miRNA targets involved in cell signaling, transcription, proliferation, immunoreactions, apoptosis and viral DNA reproduction were confirmed or predicted by informatics analysis. Conclusion： Ebv - miRNAs may be involved in the pathogenesis of NPC by regulating their target genes. Differential expression of ebv - miRNAs in NPC ceils implies the possibility of individual treatment of NPC targeting on EBV miRNAs.
出处
《现代肿瘤医学》
CAS
2010年第6期1064-1069,共6页
Journal of Modern Oncology
基金
广东省医学科研基金项目(NoB2006111)
广东省中医药局科研项目(No2008355)
