siRNA沉默LBH基因促进人支气管上皮细胞周期进展

摘要

目的:利用合成的小分子干扰RNA(siRNA)转染人支气管上皮细胞16HBE,观察LBH(limb-bud and heart) 基因表达下调对16HBE细胞周期及相关基因表达的影响.方法:脂质体2000转染靶向LBH 基因的siRNA到16HBE细胞,荧光定量RT-PCR检测siRNA转染后LBH基因表达,流式细胞术检测LBH基因沉默后16HBE 细胞周期的改变,荧光定量RT-PCR及Western blotting检测LBH基因沉默后细胞周期素E1和E2表达水平.结果:50 nmol/L siRNA转染16HBE细胞48 h后, LBH基因的表达水平只有对照组的14%;siRNA转染16HBE细胞48 h,其G1期细胞百分率比对照组减少9.28%,而S期细胞百分比增加14.08%;16HBE细胞在50 nmol/L siRNA的转染后,细胞周期素E2的表达水平为对照组的2倍,而细胞周期素E1的表达没有发生明显改变.结论:靶向于LBH基因的siRNA能有效沉默16HBE细胞中LBH基因的表达,LBH基因的表达下调促进了16HBE细胞周期G1/S期的进展;细胞周期素E2的表达上调参与了LBH沉默导致的细胞周期G1/S期的进展.%AIM : To investigate the effect of small interfering RNA( siRNA ) on limb - hud and heart( LBH )gene expression in 16HBE cells, and further to observe the change of 16HBE cell cycle after knocking down of LBH gene.METHODS: Synthetical siRNA targeting LBH gene was transfected into 16HBE cells by the method of lipofectamine 2000.The mRNA expression of LBH was examined by real time RT - PCR.The cell cycle was assayed by flow cytometry.The expression of cyclin E1 and E2 was also detected by real time RT - PCR and Western blotting.RESULTS: The expression of LBH gene decreased by 86％ in 16HBE cells transfected with 50 nmol/L of siRNA for 48 h.After transfected with siRNA for 48 h, 16HBE cells in G1 phase decreased by 9.28％ and the cells in S phase increased by 14.08％ .The expression level of cyclin E2 in 16HBE cells transfected with siRNA was twice higher than that of the negative control cells.CONCLUSION: Sequence - specific siRNA targeting LBH is capable of suppressing LBH gene expression in 16HBE cells.Down - regulation of LBH expression in 16HBE cells may promote the progress of cell cycle , and up - regulation of cyclin E2 expression plays a role in the process of G1/S phase.