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目的尾侧同源盒基因(caudal tupe homeobox gene 2,CDX2)是早期胚胎生长发育过程中重要的调节因子,近年来有研究证实CDX2基因在造血细胞增殖分化过程中亦发挥重要作用。本研究采用RNA干扰(RNA interference,RNAi)技术沉默白血病K562细胞中CDX2的表达,探讨该基因沉默前后对白血病细胞增殖和凋亡的影响,为白血病的临床治疗提供理论依据。方法设计合成针对目的基因CDX2不同编码区的3个小干扰RNA序列(CDX2-siRNA-1443,CDX2-siRNA-2009,CDX2-siRNA-921)及阴性对照序列(CDX2-siRNA-NC),将各组siRNA分别转染K562细胞,同时设未转染K562细胞组作为对照,采用实时荧光定量PCR(RT-PCR)法及蛋白质印迹法检测CDX2mRNA和蛋白的表达量变化,筛选对目的基因CDX2沉默效率最高的siRNA序列,用于下一步实验。MTT法、Annexin V-FITC/PI双染法流式细胞术检测转染前后细胞增殖和凋亡变化。结果 RT-PCR结果显示,与未转染K562细胞组相比,3组CDX2-siRNA均能有效沉默K562细胞内CDX2mRNA表达,其中CDX2-siRNA-921对CDX2mRNA沉默效率最高(66.2%),差别有统计学意义,P<0.05。蛋白质印迹法结果显示,CDX2-siRNA-921组CDX2蛋白相对表达量与未转染K562细胞组相比降低最明显(68.0%,P<0.01),该结果与RT-PCR结果相符。MTT法显示,转染CDX2-siRNA-921组细胞存活率明显低于未转染K562细胞组,而转染CDX2-siRNA-NC组细胞存活率无明显变化,提示转染CDX2-siRNA-921能够明显抑制细胞增殖。流式细胞术结果显示,与CDX2-siRNA-NC组和正常细胞组相比,转染CDX2-siRNA-921组细胞凋亡率显著增加。结论 CDX2siRNA能够有效抑制白血病细胞K562中CDX2的表达,使细胞增殖减弱、凋亡增加,有望为白血病临床治疗提供新的基因靶点和治疗方法。
The caudal tupe homeobox gene 2 (CDX2) is an important regulator of early embryonic growth and development. In recent years, studies have shown that CDX2 also plays an important role in the proliferation and differentiation of hematopoietic cells. In this study, RNA interference (RNAi) technology was used to silence the expression of CDX2 in leukemia K562 cells, and to explore the effect of the gene silencing on the proliferation and apoptosis of leukemia cells before and after leukemia, so as to provide a theoretical basis for the clinical treatment of leukemia. Methods Three small interfering RNA sequences (CDX2-siRNA-1443, CDX2-siRNA-2009, CDX2-siRNA-921) and negative control sequences (CDX2-siRNA-NC) were designed and synthesized for different coding regions of CDX2. The siRNA was transfected into K562 cells separately. At the same time, untransfected K562 cells were used as control. The expression of CDX2 mRNA and protein was detected by real-time fluorescence quantitative PCR (RT-PCR) and Western blotting. The silencing efficiency of CDX2 The highest siRNA sequence for the next experiment. MTT assay and Annexin V-FITC / PI double staining were used to detect the changes of cell proliferation and apoptosis before and after transfection. Results RT-PCR results showed that CDX2-siRNA could effectively silence CDX2 mRNA expression in K562 cells compared with untransfected K562 cells, of which CDX2-siRNA-921 had the highest efficiency of silencing CDX2 mRNA (66.2%) Statistical significance, P <0.05. The results of Western blotting showed that the relative expression of CDX2 protein in CDX2-siRNA-921 group was significantly lower than that in untransfected K562 cells (68.0%, P <0.01), which was in good agreement with RT-PCR. MTT assay showed that the viability of cells transfected with CDX2-siRNA-921 was significantly lower than that of untransfected K562 cells, but no significant changes were found in the cells transfected with CDX2-siRNA-NC, which indicated that transfected CDX2-siRNA-921 Significantly inhibited cell proliferation. Flow cytometry results showed that compared with CDX2-siRNA-NC group and normal cell group, the apoptosis rate of CDX2-siRNA-921 group was significantly increased. Conclusions CDX2 siRNA can effectively inhibit the expression of CDX2 in K562 leukemia cells and attenuate cell proliferation and increase apoptosis. It is expected to provide a new gene target and treatment for the clinical treatment of leukemia.