肿瘤相关线粒体蛋白12对移植瘤细胞增殖的影响

目的探讨肿瘤相关线粒体蛋白12(TAMP12)对小鼠移植瘤细胞增殖的影响。方法采用脂质体法将TAMP12基因及空载体转染肝癌细胞系SMMC-7721,经G418筛选获得稳定高表达TAMP12细胞株(SMMC-7721-TAMP12)及对照细胞(SMMC-7721-pcDNA3.1),实时定量PCR和蛋白印迹检测TAMP12在这两组细胞中的表达。将SMMC-7721-TAMP12和对照SMMC-7721、SMMC-7721-pcDNA3.1三组细胞分别接种于BALB/cnu/nu小鼠的前肢腋下,连续观察荷瘤小鼠中肿瘤的生长状况。接种4周后处死小鼠,取瘤块分别测量瘤体质量和体积,应用实时定量PCR和免疫组化方法检测三组移植瘤中TAMP12的表达;透射电镜观察移植瘤细胞中亚细胞器形态;并应用基因芯片技术检测三组移植瘤肿瘤细胞中的基因差异表达。结果将经实时定量PCR和蛋白印迹检测显示高表达TAMP12的SMMC-7721和对照细胞分别接种裸鼠后,三组荷瘤小鼠中肿瘤的生长曲线呈现明显差异,转染TAMP12的荷瘤小鼠中肿瘤生长速度明显快于对照组荷瘤小鼠。肉眼观察可见实验组肿瘤组织中血管较对照组丰富;电镜观察发现实验组的细胞分裂增殖明显。基因芯片显示,该基因的高表达可促进多个与细胞增殖相关的基因表达上调。结论TAMP12基因具有促进细胞增殖的作用,其机制可能与调节细胞增殖的胰岛素样受体介导的信号转导通路有关。

Effects of tumor-associated mitochondrial protein 12 on tumor cell growth in xenograft mice

Objective To explore the effects of tumor-associated mitochondrial protein 12(TAMP12) on tumor cell growth in the xenograft mice. Methods Human hepatoma cell line SMMC-7721 was stably transfected with either recombinant vector pcDNA3.1-TAMP12(SMMC-7721-TAMP12)or pcDNA3.1(SMMC-7721-pcDNA3.1)as mock control.Real-time PCR and Western blotting assay were used to detect the expression of TAMP12 in transfected cell lines.Non-transfected and two types of transfected SMMC-7721 cell lines were further injected into BALB/cnu/nu mice,and the growth rate of xenografts was continuously observed.After 4 weeks,xenograft tumors were harvested to compare the volume and weight among different groups.TAMP12 expression was parallelly detected using real-time PCR and immunohistochemistry in these tumor sections.The ultra-structures of tumor cells in situ from xenografts were observed under transmission electron microscope.Finally,TAMP12-regulated genes were screened by microarray assay in these xenograft tumor specimens. Results It was indicated by real-time PCR and Western blotting that TAMP12 was higher-expressed in SMMC-7721-TAMP12 cell lines compared to the non-transfected or mock control counterparts.The growth rate of the xenografts exhibited statistical significance among different groups.The xenografts derived from SMMC-7721-TAMP12 cell line experienced faster growth kinetics and had more blood vessel infiltration.It was demonstrated by transmission electron microscope scanning that overexpression of TAMP12 promoted mitosis and protein synthesis in SMMC-7721 cell lines.It was revealed by microarray assay that the up-regulation of TAMP12 led to the modulation of multiple gene expression,which were involved in several signaling pathways responsible for cell cycling and proliferation. Conclusion TAMP12 may possess the ability to accelerate the proliferation of cell lines in vivo,which may be mediated through the engagement of insulin-like growth factor receptor.