下调hnRNP A2/B1基因表达对骨肉瘤MG-63细胞生长的影响及其机制

Effect of down-regulation of hnRNP A2/B1 gene expression on the growth of osteosarcoma MG-63 cells and its mechanism

目的:通过下调核内不均一核糖核蛋白A2/B1(heterogeneous-nuclear ribonucleoprotein A2/B1,hn RNP A2/B1)的表达,探讨该基因对骨肉瘤MG-63细胞体内外生长的作用及其分子机制。方法:将细胞分为空白对照组(未转染)、阴性对照组(转染阴性对照质粒)和hn RNP A2/B1下调组(转染sh RNA-hn RNP A2/B1干扰质粒)。质粒转染骨肉瘤MG-63细胞后,观察绿色荧光蛋白的表达,以鉴定转染效果。采用实时荧光定量PCR和蛋白质印迹法检测3组细胞中hn RNP A2/B1、磷酸化的Akth r308(phosphorylated Akth r308,p-Akth r308)、p-AktSer473及Akt下游分子p21、p27、cleaved caspase-3和Bcl-2的表达变化;MTT法和FCM法分别检测各组细胞的增殖活力、周期分布和早期凋亡率。将阴性对照组和hn RNP A2/B1下调组细胞分别接种入同一裸鼠的对称部位皮下组织,观察移植瘤生长情况,并采用免疫组织化学法检测各组移植瘤组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和Ki-67的表达。结果:阴性对照组和hn RNP A2/B1下调组细胞中均有绿色荧光蛋白表达,说明质粒转染成功。与空白对照组和阴性对照组相比,hn RNP A2/B1下调组中hn RNP A2/B1、p-AktThr308、p-AktSer473和Bcl-2表达水平显著降低(P值均<0.05),且p21、p27和cleaved caspase-3表达水平明显升高(P值均<0.05);hn RNP A2/B1下调组细胞活力明显降低(P<0.05),细胞周期阻滞于G1期(P<0.01),S期细胞比例降低(P<0.01),同时细胞的早期凋亡率升高(P<0.001)。动物体内研究证实,hn RNP A2/B1下调组裸鼠移植瘤体积明显小于阴性对照组(P<0.05),并且hn RNP A2/B1下调组移植瘤组织中PCNA和Ki-67的表达水平明显降低(P值均<0.05)。结论:下调hn RNP A2/B1基因表达能够抑制骨肉瘤细胞的生长,其机制可能涉及Akt信号通路的调控。

Objective： To investigate the role of heterogeneous-nuclear ribonucleoprotein A2/B1 （hnRNP A2/B1） gene expression in growth of osteosarcoma MG-63 cells in vitro and in vivo by down- regulating the expression of hnRNP A2/B1 protein, and to explore its mechanism. Methods： The MG-63 cells were divided into blank control group （not transfected）, negative control group （transfected with a negative control plasmid） and hnRNP A2/B1 down- regulation group （transfected with shRNA-hnRNP A2/B1 plasmid）. After transfection with different plasmids, the expression of green fluorescent protein （GFP） in MG-63 cells was observed to identify the transfection efficacy. The expression levels of hnRNP A2/B1, phosphorylated AktThr3~8 （p-AktThr3~8）, p-Aktset473, and downstream molecules in Akt signaling pathway such as p21, p27, cleaved caspase-3 and Bcl-2 were detected by real-time fluorescent quantitative-PCR and Western blotting. The cell viability, cell cycle distribution and early apoptosis rate were detected by MTT and FCM methods, respectively. The MG-63 cells of the negative control group and hnRNP A2/B1 down- regulation group were subcutaneously implanted into symmetrical parts of the body of the same nude mice. The growth of xenografted tumors was observed. The expression level of proliferating cell nuclear antigen （PCNA） and Ki-67 in xenografted tumors were detected by immunohistochemistry. Results： The MG-63 cells in the negative control group and hnRNP A2/B1 down-regulation group expressed GFP, indicating that the plasmids were successfully transfected. As compared with the blank control group and the negative control group, the expression levels of hnRNP A2/B1, p-AktThr3~8, p-Aktset473 and Bcl-2 were significantly decreased in hnRNP A2/ B1 down-regulation group （all P 〈 0.05）, but the levels of p21, p27 and cleaved caspase-3 were significantly increased （all P 〈 0.05）. The cell viability of hnRNP A2/B1 down-regulation group was significantly lower than those of the blank control group and the negative control group （both P 〈 0.05）. The down-regulation of hnRNP A2/B1 induced cell cycle arrest at G1 phase （P 〈 0.01）, decreased the proportion of MG-63 cells in S phase （P 〈 0.01）, and increase the rate of early apoptosis （P 〈 0.001）. In vivo animal study confirmed that tumor volume of hnRNP A2/B1 down-regulation group was smaller than that of the negative control group （P 〈 0.05）, while the expression levels of PCNA and Ki-67 in tumor tissues of hnRNP A2/B1 down-regulation group were reduced （both P 〈 0.05）. Conclusion： Down-regulation of hnRNP A2/B1 of osteosarcoma, and its mechanism may be gene expression can inhibit malignant growth nvolved in regulation of Akt signaling pathway.