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目的探讨靶向干扰CIAPIN1基因表达对慢性粒细胞白血病(CML)细胞系K562粒系分化的影响。方法针对CIAPIN1基因设计并构建短发卡RNA(shRNA)真核表达载体,转染K562细胞并作为干扰组,以无关序列shRNA处理的K562细胞作为对照组。采用Real-time PCR及Western blot技术鉴定干扰效率;采用瑞氏染色和电镜分别观测K562细胞形态学和超微结构的改变;采用Real-time PCR方法检测粒系分化标志基因中性粒细胞胞浆因子1(NCF1)、血清黏蛋白1(ORM1)以及粒系分化转录因子CCAAT/增强子结合蛋白(C/EBPα)、低氧诱导因子1α(HIF1α)mRNA水平的改变;流式细胞术检测K562细胞表面分化抗原CD11b表达的改变;Western blot技术检测ERK1/2、JNK、p38MAPK及Akt磷酸化水平的改变。结果与对照组相比,干扰组的CIAPIN1基因表达被有效抑制(P<0.05);K562细胞的形态和超微结构趋向成熟粒细胞阶段;NCF1、ORM1、C/EBPα及HIF1αmRNA表达水平升高(P<0.05);CD11b表达升高(P<0.01);ERK1/2磷酸化水平升高(P<0.05),而JNK、p38MAPK及Akt磷酸化水平未见明显改变。结论抑制CIAPIN1基因表达能促进K562细胞向成熟的粒细胞阶段分化,ERK1/2磷酸化水平的升高可能参与了该分化过程。
Objective To investigate the effect of targeted interference of CIAPIN1 gene expression on the differentiation of the chronic myeloid leukemia (CML) cell line K562. Methods The short hairpin RNA (shRNA) eukaryotic expression vector was designed and constructed based on the CIAPIN1 gene. The K562 cells were transfected into K562 cells and served as the interference group. K562 cells treated with no relevant shRNA were used as the control group. Real-time PCR and Western blot were used to identify the interference efficiency. Morphological and ultrastructural changes of K562 cells were observed by Wright staining and electron microscopy. Real-time PCR was used to detect the polymorphisms of neutrophil cytoplasm (NCF1), serum mucoprotein 1 (ORM1), and the levels of C / EBPα and HIF1α mRNA were detected by flow cytometry. The expression of K562 Cell surface differentiation antigen CD11b expression changes; Western blot detection of ERK1 / 2, JNK, p38MAPK and Akt phosphorylation changes. Results Compared with the control group, CIAPIN1 gene expression was significantly inhibited in the interference group (P <0.05). The morphology and ultrastructure of K562 cells tended to mature granulocyte stage. The expression levels of NCF1, ORM1, C / EBPα and HIF1α mRNA were increased (P <0.05). The expression of CD11b was increased (P <0.01). The phosphorylation of ERK1 / 2 was increased (P <0.05), while the phosphorylation of JNK, p38MAPK and Akt did not change significantly. Conclusion Inhibition of CIAPIN1 gene expression can promote the differentiation of K562 cells to mature granulocyte stage. The increase of ERK1 / 2 phosphorylation may be involved in the differentiation process.