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目的在细胞水平观察microRNA(miRNA)载体瞬时转染293T细胞株后对KiSS1基因转录、翻译的干预效应。方法以SD大鼠基因组DNA为模板,克隆KiSS1基因全长序列,构建重组载体pcDNA3.1(+)-KiSS1。应用miRNA设计软件,针对大鼠KiSS1基因的4个不同靶位点设计4对干扰序列及1对非相关性的阴性对照序列,得到miRNA表达载体p-KiSS1-miRNA1-4及p-KiSS1-miRNA-neg。利用脂质体介导将上述载体与pcDNA3.1(+)-KiSS1共转染293T细胞,采用荧光显微镜及免疫荧光技术检测转染72 h后细胞中各载体表达。采用实时荧光定量-PCR(RT-RCR)及Western blot技术观察转染48 h及72 h后miRNA表达载体对KiSS1基因的干预效应。结果荧光显微镜观察到miRNA载体转染293T细胞后可表达绿色荧光蛋白,免疫荧光结果显示转染pcDNA3.1(+)-KiSS1载体组可见红色的KiSS1蛋白在细胞质中表达;RT-PCR结果显示干预组细胞KiSS1 mRNA表达显著低于阳性对照组和阴性对照组(Pa<0.05)。其中p-KiSS1-miRNA2组KiSS1基因表达抑制最显著,降低了85%;Western blot结果显示p-KiSS1-miRNA2干预组KiSS1蛋白表达明显低于阳性对照组及阴性对照组。结论 4个miRNA载体对大鼠KiSS1基因均具有抑制效应,其中针对KiSS1 mRNA序列304～324 bp的p-KiSS1-miRNA2抑制效果最佳,为个体水平沉默KiSS1防治性早熟的研究提供依据。
Objective To observe the effect of microRNA (miRNA) vector transient transfection on 293T cell line at the cellular level on the transcription and translation of KiSS1 gene. Methods The genomic DNA of SD rats was used as a template to clone the full - length KiSS1 gene. The recombinant plasmid pcDNA3.1 (+) - KiSS1 was constructed. Using miRNA design software, four pairs of interference sequences and one pair of non-related negative control sequences were designed for four different target sites of KiSS1 gene to obtain miRNA expression vectors p-KiSS1-miRNA1-4 and p-KiSS1-miRNA -neg. 293T cells were co-transfected with pcDNA3.1 (+) - KiSS1 by lipofectamine. The expression of each vector was detected by fluorescence microscopy and immunofluorescence assay 72 h after transfection. The effects of miRNA expression vector on KiSS1 gene expression at 48 h and 72 h after transfection were observed by real-time fluorescence quantitative-PCR (RT-PCR) and Western blot. Results Fluorescence microscopy showed that miRNA vector transfected 293T cells could express green fluorescent protein. The results of immunofluorescence showed that the transfected pcDNA3.1 (+) - KiSS1 vector showed that the red KiSS1 protein was expressed in the cytoplasm. RT-PCR results showed that intervention KiSS1 mRNA expression in the group of cells was significantly lower than that of the positive control group and the negative control group (Pa <0.05). The expression of KiSS1 in p-KiSS1-miRNA2 group was most significantly decreased by 85%. Western blot results showed that the expression of KiSS1 protein in p-KiSS1-miRNA2 group was significantly lower than that in positive control group and negative control group. Conclusions All 4 miRNA vectors have inhibitory effect on KiSS1 gene in rat. The optimal inhibitory effect of pSSI-miRNA2 against the KiSS1 mRNA sequence of 304 ~ 324 bp is the best.