RNA干扰抑制HPV16 E6基因的表达对HPV16阳性宫颈癌细胞中基因表达谱的影响（英文）

Gene expression profile in HPV16-positive human cervical carcinoma cells treated by RNA interference against HPV16 E6 gene

目的:探讨RNAi抑制E6基因的表达对宫颈癌细胞基因表达谱的影响。方法:利用脂质体将已验证有效的表达针对E6基因小发卡RNA(shRNA)的重组质粒转染到宫颈癌细胞Ca SKi中,提取宫颈癌细胞总RNA并利用Agilent Human 1A寡核苷酸表达芯片检测RNAi后Ca SKi细胞基因表达谱的变化,最后实时PCR检测CDKN2B(p15)和MKI67来验证芯片分析结果。结果:与Ca SKi/PSN相比,共有2 765个基因表达有明显差异,其中有2 709个上调基因(包括代谢相关基因、肿瘤抑制基因、信号转导相关基因、凋亡基因等)和56个下调基因(包括原癌基因、DNA结合和转录基因、代谢相关基因、信号转导相关基因、细胞周期和发育相关基因等)。实时PCR结果表明CDKN2B(p15)和MKI67的变化与基因芯片分析结果一致。结论:联合应用RNAi和基因芯片分析技术可以为研究HPV16 E6与宿主细胞相互作用分子机制提够更丰富的资料和信息,并提供新的研究策略和途径。

Objective：High risk human papillomavirus type 16 was well recognized in cervical earcinoma and viral oncogene E6 played an important role in carcinogenesis of the cervix. Our study aims to investigate that how RNAi against viral E6 gene had the impact on the gene expression profile of HPVI6 -positive cervical carcinoma cell CaSKi. Methods：The effective recombinant plasmids which express small hairpins RNA （shRNA） in vivo against E6 gene were constructed and transfected into cervical eareinonm cell CaSKi by lipofeetamine 2000 reagent. Gene expression profiles of the cells were detected with Agilent Human 1A Oligo - microarray and CDKN2B （ p15 ） and MKI67 were detected with real - time PCR method to validate the results of microarray analysis. Results ： In contrast to gene profi- ling of CaSKi/PSN ,total 2 765 differentially expressed genes were identified including 2 709 up - regulated ones con- sisting of metabolism - associated genes, tumor suppressor genes, signal transduetion - associated genes, apoptosis genes and others with unknown function and 56 down -regulated ones consisting of oncogenes, DNA -binding and transcription genes, metabolism - associated genes, signal transduction - associated genes, cell cycle and development - associated genes in CaSKi/PS6. The variations of CDKN2B （p15） , MKI67 detected by real - time PCR were in a- greement with those by microarray. ConelusionzThese findings suggested that application of RNA interference tcch- nohgy and expression mieroarray provided us more intbrmation about molecular interaction between HPV16 E6 and host eell and expanded the extent of research and provided new strategy for investigation mechanism of HPVI6 oneo- gene E6 in eervical carcinoma.