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背景与目的:RNA干扰(RNAinterference,RNAi)具有高效特异阻断基因表达的特点,在基因功能研究、信号转导通路的研究以及基因治疗中得到很广泛的应用。本实验通过体外转录合成siRNA和构建表达shRNA的质粒载体两种方法,检测鼻咽癌细胞系的RNAi作用,建立一个较完善的RNAi技术平台。方法:体外转录合成或通过pSUPER.retro构建表达针对GFP和荧光素酶的siRNAs或shRNAs,将它们和报道基因一起瞬时转染HeLa、CNE1、CNE2和5-8F细胞,用荧光显微镜和Westernblot检测GFP表达情况;用荧光素酶检测系统分析荧光素酶的活性。结果:3’末端带UU突出的siGFP2和psGFP能特异性地抑制细胞中GFP的表达,而单链反义RNA和3’末端不带UU突出的siGFP1无此作用。定量荧光素酶活性分析发现,siLuc能抑制HeLa、CNE1、CNE2和5-8F细胞中荧光素酶的表达,其抑制率分别达到91.43%,78.01%,90.30%和62.85%。HeLa细胞瞬时转染psLuc后,荧光素酶的表达抑制率可达到78.22%。结论:体外转录合成的siRNA和表达shRNA的质粒均能诱发RNAi效应。3’UU突出末端在siRNA诱发的RNAi中起作用。鼻咽癌细胞与HeLa细胞一样存在RNAi效应。
BACKGROUND & OBJECTIVE: RNA interference (RNAi) is characterized by efficient and specific blocking of gene expression. It has been widely used in gene function studies, signal transduction pathways and gene therapy. In this study, two methods of transcribing siRNA in vitro and constructing plasmid vector for shRNA expression were used to detect the RNAi effect of NPC cell lines and establish a perfect RNAi technology platform. METHODS: siRNAs or shRNAs targeting GFP and luciferase were constructed by in vitro transcriptional synthesis or by pSUPER.retro. They were transiently transfected into HeLa, CNE1, CNE2 and 5-8F cells with the reporter gene. GFP was detected by fluorescence microscopy and Western blot The luciferase assay system was used to analyze luciferase activity. Results: The siGFP2 and psGFP with UU prominence on the 3 ’end could specifically inhibit the expression of GFP in cells, while single-stranded antisense RNA and siGFP1 without UU prominence at the 3’ end did not. Quantitative luciferase activity analysis showed that siLuc could inhibit the expression of luciferase in HeLa, CNE1, CNE2 and 5-8F cells with the inhibitory rates of 91.43%, 78.01%, 90.30% and 62.85%, respectively. After transient transfection of psLuc in HeLa cells, the inhibition rate of luciferase expression reached 78.22%. CONCLUSIONS: The siRNAs synthesized in vitro and plasmids expressing shRNA all induce the RNAi effect. The 3’UU overhang plays a role in siRNA-induced RNAi. Nasopharyngeal carcinoma cells have the same RNAi effect as HeLa cells.