Yang Y, Liu W-J, Huang H-P, Guo Q-L
Department of Pediatrics, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China.
Eur Rev Med Pharmacol Sci. 2016 Jul;20(14):3101-11.
To Knockdown Homeobox A5 (HOXA5) expression by HOXA5-specific siRNA and evaluate the effects on Livin and Smac proteins expression in acute T cell leukemia Jurkat cells.
We designed and constructed HOXA5-specific siRNA, and using liposomes we transfected Jurkat cells with this siRNA. The experiment was designed for three groups: (i) experimental group with Jurkat cells transfected with HOXA5-specific siRNA (siRNA transfection group), (ii) negative control group (irrelevant siRNA transfection) with Jurkat cells transfected with pRNAT-U6.1-siD and (iii) normal control group (untransfected Jurkat cells, only with equivalent amounts of cells and medium). We used FQ-PCR and Western blot to detect the relative expression levels of HOXA5 mRNA and protein in each group separately. The Western blot was also used to detect Livin and Smac protein levels in Jurkat cells.
Expression levels of HOXA5 mRNA and protein were significantly reduced in the group with Jurkat cells transfected with HOXA5 siRNA (p<0.05). The expression of Livin protein was significantly down-regulated (p<0.05) while the expression of Smac protein was significantly up-regulated (p <0.05).
HOXA5-specific siRNA effectively silenced the HOXA5 gene expression and down-regulation of HOXA5 induced the down-regulation of Livin protein expression and up-regulation of Smac protein. We suggest the HOXA5 gene to be considered as the new target for acute leukemia gene therapy.
利用HOXA5特异性小干扰RNA（siRNA）敲低同源盒A5（HOXA5）的表达，并评估其对急性T淋巴细胞白血病Jurkat细胞中Livin和Smac蛋白表达的影响。
我们设计并构建了HOXA5特异性siRNA，并用脂质体将该siRNA转染至Jurkat细胞。实验分为三组：（i）用HOXA5特异性siRNA转染Jurkat细胞的实验组（siRNA转染组），（ii）用pRNAT-U6.1-siD转染Jurkat细胞的阴性对照组（无关siRNA转染），以及（iii）正常对照组（未转染的Jurkat细胞，仅含等量细胞和培养基）。我们分别使用荧光定量聚合酶链反应（FQ-PCR）和蛋白质免疫印迹法（Western blot）检测每组中HOXA5 mRNA和蛋白的相对表达水平。蛋白质免疫印迹法还用于检测Jurkat细胞中Livin和Smac蛋白水平。
用HOXA5 siRNA转染Jurkat细胞的组中，HOXA5 mRNA和蛋白的表达水平显著降低（p<0.05）。Livin蛋白的表达显著下调（p<0.05），而Smac蛋白的表达显著上调（p<0.05）。
HOXA5特异性siRNA有效沉默了HOXA5基因表达，HOXA5的下调诱导了Livin蛋白表达的下调和Smac蛋白的上调。我们建议将HOXA5基因视为急性白血病基因治疗的新靶点。
