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目的探讨慢病毒介导的短发夹RNA(shRNA)敲低NB4急性早幼粒细胞白血病(APL)细胞维甲酸诱导基因Ⅰ(RIG-Ⅰ)对全反式维甲酸(ATRA)诱导NB4细胞分化的影响及其机制。方法将含有人RIG-Ⅰ基因shRNA序列(RIG-Ⅰ-shRNA)及含绿色荧光蛋白(GFP)基因的慢病毒感染NB4细胞,实时定量PCR(qRT-PCR)及Western blot法分别检测ATRA诱导前后NB4细胞RIG-Ⅰ mRNA和蛋白水平;流式细胞术观察慢病毒感染效率和RIG-Ⅰ-shRNA对ATRA诱导NB4细胞分化的影响;Western blot法检测敲低RIG-Ⅰ水平后,ATRA处理对蛋白激酶B308位苏氨酸(AKT-Thr308)磷酸化的调控效应。结果含RIG-Ⅰ-shRNA序列的慢病毒可成功感染NB4细胞,下调ATRA诱导的RIG-Ⅰ水平;敲低RIG-Ⅰ后,明显抑制ATRA诱导NB4细胞分化,同时上调AKT-Thr308磷酸化水平。结论敲低NB4细胞RIG-Ⅰ蛋白可上调AKT-Thr308磷酸化水平抑制ATRA诱导的急性早幼粒白血病细胞分化。
Objective To investigate the effect of RIG-Ⅰ on the differentiation of NB4 cells induced by all-trans retinoic acid (ATRA) induced by lentivirus-mediated short hairpin RNA (shRNA) knockdown in NB4 cell line of acute promyelocytic leukemia (APL) Influence and Mechanism. Methods The lentivirus containing RIG-Ⅰ-shRNA and GFP containing human RIG-Ⅰ gene were transfected into NB4 cells. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression of ATRA The mRNA and protein levels of RIG-Ⅰ in NB4 cells were detected by flow cytometry. The effect of lentivirus infection and the effect of RIG-Ⅰ-shRNA on the differentiation of NB4 cells induced by ATRA were observed by flow cytometry. After knockdown of RIG-Ⅰ by Western blot, Regulation of phosphorylation of kinase B308 threonine (AKT-Thr308). Results The lentivirus with RIG-Ⅰ-shRNA sequence could successfully infect NB4 cells and down-regulate the ATRA-induced RIG-Ⅰ level. After knockdown of RIG-Ⅰ, ATRA-induced NB4 cell differentiation was significantly inhibited and AKT-Thr308 phosphorylation was also up-regulated. Conclusion Knockdown of RIG-Ⅰ protein in NB4 cells can up-regulate AKT-Thr308 phosphorylation and inhibit ATRA-induced acute promyelocytic leukemia cell differentiation.