脑糖原磷酸化酶小干扰RNA对人骨肉瘤细胞MG63增殖的抑制作

Brain type glycogen phosphorylase small interfering RNA inhibits proliferation of human osteosarcoma cell lines

目的观察脑糖原磷酸化酶（PYGB）对人骨肉瘤细胞株MG63的细胞增殖、细胞凋亡、细胞周期分布的影响。方法通过实时定量聚合酶链反应（Real-time PCR）方法检测35例骨肉瘤和15例骨囊肿标本的PYGB表达水平。采用Western blot法挑选PYGB高表达的骨肉瘤细胞株。采用细胞计数试剂盒（CCK-8）方法测定细胞增殖，使用流式细胞仪检测MG63细胞凋亡和细胞周期，Western blot法分别检测细胞凋亡相关蛋白B细胞淋巴瘤/白血病-2相关X蛋白（bax）和B细胞淋巴瘤/白血病-2（bcl-2）的表达。结果骨肉瘤组的PYGB表达（0.105±0.945）水平明显较骨囊肿组（0.062±0.038）高（P＝0.006）。小干扰RNA沉默PYGB后的骨肉瘤细胞的增殖明显降低，PYGB干扰组的G1期细胞比例从（49.74±1.54）%增加至（69.41±2.35）%（P＝0.004），而S期细胞比例从（21.04±2.59）%下降至（14.15±0.98）%（P＝0.028）。G2期百分比从（25.62±0.64）%减少至（8.19±0.11）%（P＝0.002），表明细胞周期受阻断在G1期。PYGB干扰组的bax的表达水平显著增加，而bcl-2的表达水平显著降低。结论PYGB的小干扰RNA通过靶向抑制半胱氨酰天冬氨酸特异性蛋白酶（Caspase）/bcl和细胞周期蛋白依赖激酶1（CDK1）信号通路而抑制人骨肉瘤细胞MG63细胞的增殖。

Objective To observe the effect of brain type glycogen phosphorylase （PYGB） small interfering RNA （siRNA） on proliferation, apoptosis, distribution of cell cycle in human osteosareoma cell line MG63 with significant expression level of PYGB. Methods Thirty - five samples of osteosareoma and 15 samples of bone cysts were detected for the PYGB expression level using real - time quantitative poly- merase chain reaction（ Real- time PCR）. Western blotting was used to select osteosarcoma cell lines with high expression level of PYGB. MG63 cells were taken out for the following study. Cell counting kit - 8 （CCK- 8） assay was preformed to evaluate the cell proliferation. Cell apoptosis and cell cycle of MG63 cells were identified using flow cytometry. Besides, cell invasion and migration were evaluated by Transwell assay. In addition, cell apoptosis and metastasis related proteins B cell lymphoma/leukemia -2 associated X protein （ bax）, B cell lymphoma/leukemia - 2 （ bcl - 2 ） were measured by Western blotting. Results Expression of PYGB exhibited a higher level in osteosarcoma samples （0. 105 - 0. 945 ） than that in bone cyst （0. 062± 0. 038, P = 0. 006 ）. The cell proliferation of MG63 ceils were inhibited by PYGB siRNA. PYGB knockdown in MG63 cells increased the proportion of G1 phase cells from （ 49. 74 ±1.54 ） % to （69. 41±2. 35 ） % （ P = 0.004 ）, while decreased the proportion of S phase cells from （21.04±2. 59 ） % to （ 14. 15 ± 0. 98 ） % （ P = 0. 028 ） and decreased the proportion of G2 phase cells from （ 25.62 ± 0. 64 ） % to （8.19 ± 0. 11 ）% （P = 0.002 ）. The expression of bax significantly reduced in PYGB siRNA group while the expression of bcl - 2 significantly reduced in PYGB siRNA group. Low expression of PYGB reduced G1 phase of the cell cycle transition. Conclusion PYGB siRNA inhibited proliferation of MG63 cells by targeting cysteinyl aspartate- specific protease/bcl and cyclin- dependent kinase （CDK）1 signa- ling pathway.