靶向高迁移率族蛋白1基因的高效RNA干扰序列的筛选

Screening of high effective siRNAs targeting high mobility group box 1 protein gene

目的筛选靶向晚期炎症因子高迁移率族蛋白1(High mobility group box 1 protein,HMGB1)基因的高效RNA干扰(siRNA)序列,并检测其体外生物学效应。方法选择针对HMGB1基因mRNA的3条不同序列,用T7体外转录系统合成3条siRNA,转染RAW264.7细胞,6 h后,加入500 ng/ml脂多糖(Lipopolysaccharide,LPS)分别刺激24、48 h,另设单纯LPS刺激组和只加培养液的对照组。采用RT-PCR检测各组细胞中HMGB1基因mRNA的转录水平,ELISA检测细胞培养上清液中HMGB1的含量。结果体外合成的HMGB1 siRNA1～3经琼脂糖凝胶电泳分析,均可见21 bp的RNA片段;经LPS刺激24和48 h,RAW264.7细胞中HMGB1基因mRMA的转录水平和细胞培养上清液中HMGB1的含量与对照组相比均显著升高,3条HMGB1 siRNA均能抑制细胞中HMGB1基因mRNA的转录水平,并减少细胞培养上清液中HMGB1的含量,其中以HMGB1siRNA1的效果最为明显。结论 LPS可刺激RAW264.7细胞释放大量HMGB1,同时增加细胞中mRNA的转录水平;HMGB1siRNA能有效降低HMGB1蛋白和mRNA水平,有望成为控制晚期炎症发展的新的治疗手段。

Objective To screen the high effective siRNAs targeting the gene encoding high mobility group box protein 1 （HMGB1）, a late mediator of inflammation, and determine its biological effect in vitro. Methods Three siRNA sequences specific to HMGB1 mRNA were selected and synthesized by T＇] transcription system in vitro. RAW264. 7 cells were divided into five groups. The cells in intervention groups 1, 2 and 3 were transfected with synthesized HMGB1 siRNAs 1 N3 respectively and, 6 h later, stimulated with 500 ng/ml lipopolysaccharide （LPS） for 24 and 48 h, while those in LPS group were untransfected and only stimulated with LPS, and those in control group were only added with medium. The transcription levels of HMGB1 mRNAs in cells of various groups were determined by RT-PCR, while the HMGB1 contents in culture supernatants by ELISA. Results The RNA fragments each at a length of 21 bp were observed on agarose gel electrophoretic profile of synthesized HMGB1 siRNAs 1 N 3. After stimulation with LPS for 24 and 48 h, both the transcription levels of HMGB1 mRNAs and HMGB1 contents in culture supernatants of cells in three intervention groups were significantly higher than those in control group. All the three HMGB1 siRNAs inhibited the transcription of HMGB1 mRNA in cells and decreased the HMGB1 content in cell culture supernatant, of which siRNA1 showed satisfactory effect as compared with siRNA2 and siRNA3. Conclusion LPS stimulated RAW264. 7 cells to release a large quantity of HMGB1 and increased the transcription level of mRNA in the cells. HMGB1 siRNA decreased the protein and mRNA levels of HMGB1, which might provide a new method for controlling the progress of late inflammation.