Ren C, Li X, Li X, Xie Y, Fu H, Yan Z, Zhu Y
1 Institute of Clinical Laboratory Science, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People's Republic China.
2 School of medicine, Hunan normal University, Changsha 410013, PRC.
Hum Exp Toxicol. 2018 Feb;37(2):196-204. doi: 10.1177/0960327117692132. Epub 2017 Feb 23.
RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.
RNA干扰（RNAi）是一种使基因表达沉默或降至最低的强大工具，而体外腭培养是研究腭发育的一项重要技术。我们之前的研究表明，在胚胎发育过程中，全反式维甲酸诱导的腭裂（CP）小鼠模型中葡萄糖调节蛋白78（Grp78）的基因表达下调。为了探究Grp78的作用，通过显微注射器注射携带该基因小干扰RNA（siRNA）的荧光载体，将约30 pmol的siRNA注射到癌症研究所（ICR）小鼠的腭外植体中。在6、12、24、48和72小时后，将这些腭外植体从培养物中取出，观察其荧光和阿尔新蓝染色表型，并检测未折叠蛋白反应（UPR）关键成员（Grp78、肌醇反应酶1、蛋白激酶RNA样内质网激酶、激活转录因子6和X盒结合蛋白1）的表达。培养72小时后，在对照siRNA组中观察到双侧腭部分或完全融合，而在Grp78 siRNA组中发现了腭裂。在Grp78 siRNA组中，每个时间点UPR关键基因的相对mRNA丰度均低于对照siRNA组，其蛋白表达也呈现相同变化。通过小鼠腭培养的RNAi策略系统，我们发现Grp78的siRNA干扰了体外培养的小鼠腭的融合。
