Liu Jian, Sun Zhao-Hui, Shi Yu-Ling, Wang Shu-Yan, Wei Min
Department of Medical Administration, General Hospital of Guangzhou Military Area, Guangzhou 510010, Guangdong Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2010 Feb;18(1):40-4.
The objective of this study was to establish an RNAi approach that can specifically target aqp1 gene sequence in vitro, and to assess the inhibitory effect of this siRNA on K562 cells. The siRNA targeting aqp1-mRNA was designed and transfected into K562 cells by using Lipofectamine(TM) 2000 reagent. Phase-contrast microscopy was used to analyze morphology changes of K562 cells. Cell viability was determined by MTT assay, and flow cytometry and DNA ladder analysis were carried out to identify siRNA-induced apoptosis. The expression levels of aqp1-mRNA at different transfection time were detected by RT-PCR. The results showed that the siRNA was successful by established. The transfected K562 cells displayed the significant apoptosis. The aqp1-siRNAs could obviously inhibit the activity of K562 cells. Cellular DNA fragmentation was observed in the siRNA group after transfection for 48 hours, the apoptosis rates at 24, 48 and 72 hours after transfection were 24.2%, 36.1% and 42.9% respectively. The aqp1-mRNA expression in the cells treated by aqp1-siRNA for 24, 48 and 72 hours were significantly reduced by 33%, 46% and 57% respectively. It is concluded that the aqp1-siRNA can efficiently and specifically inhibited the proliferation and inducing apoptosis of K562 cells. Gene aqp1 can be a potential target point for therapy of malignant tumor.
本研究的目的是建立一种能够在体外特异性靶向水通道蛋白1（aqp1）基因序列的RNA干扰方法，并评估该小干扰RNA（siRNA）对K562细胞的抑制作用。设计靶向aqp1 - mRNA的siRNA，并使用脂质体（TM）2000试剂将其转染到K562细胞中。利用相差显微镜分析K562细胞的形态变化。通过MTT法测定细胞活力，并进行流式细胞术和DNA梯状分析以鉴定siRNA诱导的细胞凋亡。通过逆转录 - 聚合酶链反应（RT - PCR）检测不同转染时间的aqp1 - mRNA表达水平。结果表明，所建立的siRNA是成功的。转染后的K562细胞显示出明显的凋亡。aqp1 - siRNAs能够明显抑制K562细胞的活性。转染48小时后，在siRNA组中观察到细胞DNA片段化，转染后24、48和72小时的凋亡率分别为24.2%、36.1%和42.9%。用aqp1 - siRNA处理24、48和72小时的细胞中，aqp1 - mRNA表达分别显著降低33%、46%和57%。结论是，aqp1 - siRNA能够有效且特异性地抑制K562细胞的增殖并诱导其凋亡。基因aqp1可能是恶性肿瘤治疗的潜在靶点。
