论文部分内容阅读
目的:探讨肿瘤相关抗原线粒体蛋白质12基因(tumor-associated antigen mitochondrial protein 12 gene,TAMP12)表达变化对人宫颈癌HeLa细胞的抑制作用。方法:利用计算机辅助设计并化学合成3对针对TAMP12的siRNA片段,通过脂质体法将siRNA转染于TAMP12高表达的HeLa细胞中,筛选有效的siRNA片段。以RT-PCR、实时定量PCR及流式细胞术(FCM)检测肿瘤细胞TAMP12 mRNA及蛋白表达的变化。以激光扫描共聚焦显微镜(confocal lyser scarning microscope,CLSM)观察双色荧光标记蛋白的亚细胞定位和定量表达。以3H-TdR掺入实验和FCM检测阻断TAMP12基因表达对HeLa细胞增殖和凋亡的影响。结果:CLSM检测显示TAMP12蛋白主要表达在HeLa细胞线粒体中。转染siRNA-TAMP12的肿瘤细胞中TAMP12 mRNA及蛋白的表达显著降低,与对照组对比,抑制率分别为81%和87%(P<0.01);转染细胞的DNA合成受抑制,抑制率达42%(P<0.01);转染siRNA-TAMP12肿瘤细胞的凋亡率由对照细胞的8.14%上升到15.59%(P<0.01)。结论:RNA干扰可阻断TAMP12基因的表达,从而对人宫颈癌Hela细胞产生抑制作用。
Objective: To investigate the inhibitory effect of tumor-associated antigen mitochondrial protein 12 gene (TAMP12) on human cervical carcinoma HeLa cells. METHODS: Three pairs of siRNA fragments targeting TAMP12 were designed and synthesized by computer. SiRNA was transfected into HeLa cells with high expression of TAMP12 by lipofectamine to screen for effective siRNA fragments. The changes of TAMP12 mRNA and protein expression in tumor cells were detected by RT-PCR, real-time quantitative PCR and flow cytometry (FCM). Subcellular localization and quantitative expression of two-color fluorescently labeled proteins were observed with a confocal lyser sc microscopy (CLSM). The effects of T-incorporation of 3H-TdR and FCM on the proliferation and apoptosis of HeLa cells were investigated by blocking TAMP12 gene expression. Results: CLSM assay showed that TAMP12 protein was mainly expressed in HeLa mitochondria. TAMP12 mRNA and protein expression were significantly decreased in siRNA-TAMP12-transfected cells compared with the control group (81% and 87%, respectively) (P <0.01). The DNA synthesis of transfected cells was inhibited and the inhibitory rate was 42% (P <0.01). The apoptosis rate of siRNA-TAMP12 transfected cells increased from 8.14% to 15.59% (P <0.01). Conclusion: RNA interference can block the expression of TAMP12 gene and inhibit the growth of human cervical cancer Hela cells.