RNAi靶向沉默CDX2基因对白血病细胞生物学行为的影响及其作用机制  

Effects of RNAi targeting CDX2 gene expression on biological characters of human leukemia cells and its relevant mechanisms

作　　者：孙维梅[1] 李建厂[1] 贾秀红[1] 李有杰[2] 唐慎华[1] 

机构地区：[1]滨州医学院附属医院儿科,山东滨州256603 [2]滨州医学院肿瘤分子生物学重点实验室

出　　处：《天津医药》2016年第6期679-683,共5页Tianjin Medical Journal

基　　金：山东省医药卫生科技发展计划(2014WS0183);山东省自然科学基金(ZR2014HL032)

摘　　要：目的探讨小分子干扰RNA(si RNA)靶向沉默CDX2基因后对白血病细胞K562增殖、凋亡的影响,并检测BCR-ABL及相关凋亡蛋白表达量的变化,探讨其可能的作用机制。方法根据前期实验结果,成功筛选出能够特异性有效靶向沉默CDX2基因的si RNA序列(CDX2-si RNA)和相应的阴性对照序列(CDX2-si RNA-NC),应用罗氏X-treme GENE HP DNA Transfection Reagent转染K562细胞,流式细胞术检测CDX2沉默表达后对K562细胞凋亡的影响;RT-PCR法和Western Blot法分别检测BCR-ABL、Caspase-9、Bax m RNA和蛋白表达量变化。结果 MTT及流式细胞术检测显示,CDX2沉默表达后,K562细胞增殖能力减弱,凋亡率明显增加;RT-PCR和Western Blot显示,与正常细胞组和CDX2-si RNA-NC组相比,CDX2沉默表达后,CDX2-si RNA组的BCR-ABL m RNA和蛋白表达量明显降低,Caspase-9、Bax m RNA和蛋白表达量明显升高(均P<0.05)。结论 CDX2-si RNA能够明显促进白血病细胞K562凋亡,其机制可能与其抑制BCR-ABL表达,增强Caspase-9、Bax的活性有关。Objective To detect the effects of siRNA targeting CDX2 gene expression on of BCR-ABL, caspase and Bax expressions, and the mechanisms thereof. Methods According to the earlier experiments, siRNA specifically targeting CDX2 gene (CDX2-siRNA) and the negative control sequence (CDX2-siRNA-NC) were selected, and then were transfected into K562 cells by Roche X-tremeGENE HP DNA Transfection Reagent. The flow cytometry analysis was used to detect the effects of siRNA on cell apoptosis. The expressions of BCR-ABL, caspase-9, Bax mRNA and protein were tested by RT-PCR and Western blot assay. Results MTT and flow cytometry analysis showed that after the silence of CDX2 gene expression, the proliferation of K562 cells was prohibited and the apoptotic rate of K562 cells was distinctly increased compared with that of normal cell group, but the negative control group had no significant change. According to the RT-PCR and Western blot assay, in comparison with the normal cell group and the negative control group, the expression levels of BCR-ABL mRNA and protein were obviously decreased, and the difference was statistic significance. On the other hand, the expressions of caspase-9 and Bax mRNA and protein were significantly higher than those of other two groups (P<0.05). Conclusion CDX2-siRNA can promote apoptosis of K562 cells obviously, and the mechanism is related with the down-regulation of BCR-ABL and the up-regulation of caspase-9 and Bax.

关 键 词：白血病 RNA干扰 基因沉默 细胞凋亡 融合蛋白质类 BCR-ABL CDX2 RNA干扰 半胱氨酸天冬氨酸蛋白酶-9 Bax 

分 类 号：R733.7[医药卫生—肿瘤]