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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: To investigate the effect of RNAi on the gene expression profile of cervical cancer cells by inhibiting the expression of E6 gene. METHODS: Liposomes were used to transfect the recombinant plasmid of E6 gene shRNA into Ca SKi of cervical cancer cells. The total RNA of cervical cancer cells was extracted and expressed using Agilent Human 1A oligonucleotide Chips were used to detect the changes of gene expression profiles of Ca SKi cells after RNAi. Finally, real-time PCR detection of CDKN2B (p15) and MKI67 was used to verify the results of the chip analysis. Results: Compared with Ca SKi / PSN, a total of 2 765 genes were significantly different, including 2 709 up-regulated genes (including metabolism related genes, tumor suppressor genes, signal transduction related genes and apoptosis genes) Down-regulated genes (including proto-oncogenes, DNA-binding and transcriptional genes, metabolism-related genes, signal transduction related genes, cell cycle and developmental related genes, etc.) Real-time PCR results showed that the changes of CDKN2B (p15) and MKI67 were consistent with the results of the gene chip analysis. Conclusion: The combined application of RNAi and gene chip analysis technology can provide more information and information for studying the molecular mechanism of HPV16 E6 interaction with host cells, and provide new research strategies and approaches.