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目的探讨RNA干扰骨肉瘤中抗增殖蛋白(prohibitin)基因的表达对骨肉瘤细胞增殖和凋亡的影响和机制。方法收集2013年6月至2014年8月行骨肉瘤穿刺和活检存档的骨肉瘤及相应瘤旁组织的蜡块标本共40例,提取骨肉瘤及相应瘤旁组织中的总RNA及总蛋白,应用实时定量聚合酶链反应(qRT-PCR)和蛋白印迹法(Western blot)分别检测prohibitin基因的mRNA和蛋白表达;阴性对照小干扰RNA(NC-siRNA)组和RNA干扰(prohibitin-siRNA)组转染MG63细胞,不经任何处理的细胞作为空白对照组;转染48 h后提取细胞中的蛋白,Western blot检测各组细胞中prohibitin的蛋白表达以及增殖细胞核抗原(PCNA)、活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(Cleaved caspase-3)、磷脂酰肌醇3-激酶(PI3K)、丝氨酸苏氨酸激酶(AKT)、磷酸化AKT(p-AKT)的蛋白表达;CCK8实验检测并计算细胞增殖率;采用Annexin V-FITC/PI双染色法检测细胞凋亡,流式细胞术测定细胞凋亡率。结果患者骨肉瘤组织中prohibitin的mRNA和蛋白的相对表达量均显著高于瘤旁组织(P均<0.01)。与空白对照组及NC-siRNA组比较,在RNA干扰组转染prohibitin-siRNA后MG63细胞的prohibitin蛋白相对表达量显著降低(P均<0.01);RNA干扰组细胞存活率及Ki67、PCNA、PI3K、p-AKT蛋白相对表达量显著降低(P均<0.01),细胞凋亡率及Cleaved caspase-3蛋白相对表达量显著升高(P均<0.01)。结论 RNA干扰沉默prohibitin的表达可显著降低骨肉瘤细胞的增殖活力,并诱导细胞凋亡,其机制与抑制PI3K/AKT信号通路有关。
OBJECTIVE: To investigate the effect of anti-proliferative (prohibitin) gene on the proliferation and apoptosis of osteosarcoma cells induced by RNA interference in osteosarcoma. Methods A total of 40 specimens of osteosarcoma and its adjacent paraffin tissues were collected from June 2013 to August 2014 for osteosarcoma biopsy and biopsy. Total RNA and total protein were extracted from osteosarcoma and corresponding tumor tissues. The mRNA and protein expression of prohibitin gene were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot respectively. Negative control small interfering RNA (NC-siRNA) group and prohibitin-siRNA group The transfected MG63 cells without any treatment were used as blank control group. The proteins in the cells were extracted 48 h after transfection, the expressions of prohibitin protein and proliferating cell nuclear antigen (PCNA) in each group were detected by Western blot, Cystine cleaved caspase-3, phosphatidylinositol 3-kinase (PI3K), serine threonine kinase (AKT), phosphorylated AKT (p-AKT) The cell proliferation rate was detected by CCK8 assay. Apoptosis was detected by Annexin V-FITC / PI double staining and the apoptosis rate by flow cytometry. Results The relative mRNA and protein expressions of prohibitin in osteosarcoma tissues were significantly higher than those in para-tumor tissues (all P <0.01). Compared with the blank control group and the NC-siRNA group, the relative expression of prohibitin protein in MG63 cells was significantly decreased after transfection with prohibitin-siRNA in RNA interference group (all P <0.01); The survival rate of cells in RNA interference group and the expression of Ki67, PCNA, PI3K (P <0.01). The apoptosis rate and the relative expression of Cleaved caspase-3 protein were significantly increased (all P <0.01). Conclusion RNAi silence prohibitin expression can significantly reduce the proliferation of osteosarcoma cells and induce apoptosis, the mechanism of which is related to the inhibition of PI3K / AKT signaling pathway.