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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 effect of this gene on the growth of human osteosarcoma MG-63 cells in vitro and in vivo and its related molecular mechanisms by downregulating the expression of heterogeneous nuclear ribonucleoprotein A2 / B1 (hn RNP A2 / B1) mechanism. Methods: The cells were divided into blank control group (untransfected), negative control group (transfected negative control plasmid) and hn RNP A2 / B1 down-regulated group (transfection sh RNA-hn RNP A2 / B1 interference plasmid). The plasmid was transfected into osteosarcoma MG-63 cells and the expression of green fluorescent protein (GFP) was observed to confirm the transfection efficiency. The expressions of hn RNP A2 / B1, phosphorylated Akt308, p-AktSer473 and Akt downstream molecules p21, p27 and cleaved caspase-3 were detected by real-time fluorescence quantitative PCR and Western blotting. 3 and Bcl-2 were detected by MTT assay and FCM respectively. The viability, cell cycle distribution and early apoptosis rate of each group were detected by MTT and FCM respectively. The negative control group and the hn RNP A2 / B1 downregulation group were inoculated into the subcutaneous tissue of the symmetrical part of the same nude mouse respectively to observe the growth of the transplanted tumor. Immunohistochemistry was used to detect the proliferating cell nuclear antigen, PCNA) and Ki-67 expression. Results: The expression of green fluorescent protein in both negative control group and hn RNP A2 / B1 down-regulated group showed that plasmid transfection was successful. Compared with the blank control group and the negative control group, the expressions of hnRNP A2 / B1, p-AktThr308, p-AktSer473 and Bcl-2 were significantly decreased in hn RNP A2 / B1 downregulation group , P27 and cleaved caspase-3 were significantly increased (all P <0.05); the viability of hn RNP A2 / B1 down-regulated group was significantly lower than that in G1 phase (P <0.01) The proportion of S phase cells decreased (P <0.01), and the early apoptosis rate of cells increased (P <0.001). Animal studies in vivo confirmed that the xenograft tumor volume in nude mice with hn RNP A2 / B1 downregulation was significantly less than that in the negative control (P <0.05), and the expression of PCNA and Ki-67 in hn RNP A2 / B1 downregulation group was significantly decreased (P <0.05). Conclusion: Down-regulation of hn RNP A2 / B1 gene expression can inhibit the growth of osteosarcoma cells, and its mechanism may be involved in the regulation of Akt signaling pathway.