RNA干扰靶向cyclin D1基因对K562细胞化疗增敏的研究

Chemosensitization of Human Chronic Leukemia Cell Line K562 by Inhibiting the Expression of Cyclin D1 through RNA Interference

目的cyclinD1基因在细胞周期调控中起关键性作用。研究表明其过表达与肿瘤的发生、发展及预后密切相关；并且与肿瘤细胞对化疗药物抗拒性有关。抑制CYClinD1蛋白表达可达到化疗增敏作用。本研究利用RNA干扰（RNAi）技术体外观测其对K562细胞cyclinD1基因的沉默效应及增强阿霉素（ADM）对K562细胞毒性作用。方法体外构建靶向cyclinD1基因的小发夹RNA（shRNA）表达质粒，通过壳聚糖介导转染K562细胞，Westernblot分析检测转染前后CyclinD1蛋白表达变化，流式细胞仪检测细胞周期分布及凋亡率的影响以及MTT法检测K562细胞对ADM敏感性的变化。结果构建靶向cyclinD1基因的ShRNA表达质粒（pshRNA-419和PshRNA-575）经壳聚糖转染后，能显著抑制cyclinD1基因表达；影响K562细胞周期分布，诱导细胞凋亡，并降低ADM半数抑制浓度，提高了化疗敏感性。而设计一碱基突变的序列所构建的质粒并无上述生物学效应。结论沉默K562细胞cyclinD1基因表达可达到有效的化疗增敏目的。

Objective Cyclin D1 gene plays a significant role in regulating cell cycle progression. It is reported that Over-expression of cyclin D1 gene is intimately associated with origination, development and prognosis of tumor and is associated with tumor cells resistance to chemotherapy drug. Suppression of cyclin D1 protein expression leads to cellular chemosensitization. This study was to determine whether this effect also exists in chronic leukemia cell line K562 by inhibiting the expression of Cyclin D1 protein through RNA interference. Methods Plasmid vectors expressing small hairpin RNA （shRNA） targeting at cyclin D1 gene were constructed and transfected into K562 cells by chitosan. Cyclin D1 protein was examined using Western blot analysis. The cell cycle and apoptosis were determined by flow cytometry. Cellular chemosensitization was evaluated by MTT assay. Results Expression of cyclin D1 protein was markedly down-regulated after transfection with pshRNA-419 and pshRNA-575 at 48h. Down-regulation of cyclin D1 protein could effect on the redistribution of cell cycle, and induce apoptosis of K562 cells, and decrease 50% inhibitory concentration （IC50） of adriamycin and enhance cellular chemosensitization. But there had no above biological effects observed after transfection with blank vector and control vector of m-pshRNA-790 at 48h. Conclusion K562 cells could be chemosensitization by the downregulation of Cyclin D1 expression through RNA interference.