小干扰RNA沉默hPTTG1基因对卵巢癌细胞增殖和凋亡的影响

Effect of hPTTG1 siRNA on proliferation and apoptosis of ovarian cancer cells

目的:利用RNA干扰技术沉默人垂体瘤转化基因1(hPTTG1)表达,观察卵巢癌细胞增殖能力和细胞凋亡的改变并探讨分子机制。方法:化学合成靶定hPTTG1的小干扰RNA(siRNA)转染体外培养的A2780细胞,RT-PCR和Western blotting检测hPTTG1及c-myc表达水平;MTT法和[3H]-TdR掺入实验检测hPTTG1对细胞增殖的影响;annexin V/PI染色流式细胞术和TUNEL法测定各组细胞凋亡情况。结果:hPTTG1siRNA转染组hPTTG1mRNA和蛋白表达下降,抑制率分别为70.5%±3.9%和63.8%±4.5%;转染hPTTG1siRNA 24 h细胞吸光度值开始下降,48 h抑制效果最明显,抑制率为42.9%±5.2%;转染hPTTG1siRNA后细胞[3H]-TdR放射性计数明显低于空白组和阴性对照组;hPTTG1siRNA干扰组细胞存活率下降,细胞凋亡率和坏死率均增加;TUNEL染色分析结果可见hPTTG1siRNA干扰组凋亡指数明显高于空白组和阴性对照组;hPTTG1干扰后c-mycmRNA和蛋白表达均下调。结论:hPTTG1siRNA通过下调c-myc表达抑制A2780细胞增殖,诱导细胞凋亡,hPTTG1可作为卵巢癌基因治疗的候选靶点。

AIM：To observe the proliferation and apoptosis of ovarian cancer cells by silencing the expression of human pituitary tumor-transforming gene 1（hPTTG1） using RNA interference technique.METHODS：The chemically synthesized siRNA targeting hPTTG1 was transfected into ovarian cancer cell line A2780 in vitro.The expression levels of hPTTG1 and c-myc were examined by RT-PCR and Western blotting.Cell proliferation was measured by MTT colorimetric assay and -TdR incorporation test.Cell apoptosis was detected by flow cytometry with annexin V/PI and TUNEL labeling.RESULTS：The expression of hPTTG1 at mRNA and protein levels was inhibited after transfection of hPTTG1 siRNA.The inhibitory efficiency was 70.5%±3.9% and 63.8%±4.5%,respectively.The absorbance began to decrease 24 h after transfection of hPTTG1 siRNA,and the highest inhibitory rate was 42.9%±5.2% at 48 h post-transfection.Radioactive incorporation of -TdR in hPTTG1 siRNA group was lower than that in normal and negative groups.The survival rate declined while the apoptotic rate and necrotic rate increased in hPTTG1 siRNA group.Apoptotic index in hPTTG1 siRNA group was higher than that in normal and negative groups.The expression of c-myc at mRNA and protein levels was down-regulated.CONCLUSION：Cell proliferation is inhibited and cell apoptosis is induced by hPTTG1 siRNA through down-regulating the expression of c-myc.hPTTG1 can be regarded as a candidate gene for ovarian cancer gene therapy.