调控Pokemon-p14ARF-p53通路对人结肠癌细胞增殖和凋亡的影响  

Effect of Regulating Pokemon-pl4ARF-p53 Pathway on Proliferation and Apoptosis of Human Colon Cancer Cells

作　　者：朱迎春[1] 徐凌[2] 王锋[2] 郭传勇[2] 柏乃运[3] 陈岳祥[1] 

机构地区：[1]第二军医大学附属长征医院消化内科,200003 [2]同济大学附属第十人民医院消化内科 [3]上海市闸北区中心医院消化内科

出　　处：《胃肠病学》2013年第2期71-75,共5页Chinese Journal of Gastroenterology

摘　　要：背景:研究发现转录抑制因子Pokemon是一种关键性致癌因子,在多种人类恶性肿瘤中表达异常上调,在体内、外实验中均能促进细胞的肿瘤性转化。Pokemon对抑癌基因ARF具有特异性转录抑制作用。目的:应用RNA干扰技术抑制人结肠癌细胞株HT-29的Pokemon表达,观察其表达抑制对肿瘤细胞增殖和凋亡的影响并探讨其可能的分子机制。方法:根据Pokemon cDNA序列设计干扰序列,构建重组干扰质粒,经脂质体介导转染入HT-29细胞。以real time RT-PCR和蛋白质印迹法检测Pokemon、p14ARF、p53表达,流式细胞术检测细胞周期和细胞凋亡。结果:Pokemon siRNA能有效抑制HT-29细胞中的Pokemon mRNA和蛋白表达,mRNA相对表达量为0.29±0.04,同时p14ARF、p53 mRNA和蛋白表达明显上调,mRNA相对表达量分别为3.03±0.49和2.80±0.25。与转染无效序列siRNA的HT-29细胞相比,Pokemon表达抑制的HT-29细胞G0/G1期细胞比例增加(43.6%±2.3%对34.7%±1.9%,P<0.05),细胞凋亡增多(10.7%±1.9%对2.7%±0.4%,P<0.05)。结论:人结肠癌细胞中的Pokemon表达与p14ARF、p53表达之间存在负相关关系,抑制Pokemon可通过上调p14ARF-p53信号通路阻滞肿瘤细胞的细胞周期进程,并诱导细胞凋亡。调控Pokemon-p14ARF-p53信号通路有望作为结肠癌的治疗靶点。Background： Transcriptional repressor Pokemon was identified as a critical factor in oncogenesis. It is aberrantly overexpressed in many human cancers, and leads to overt oneogenic transformation in both in vitro and in vivo models. Pokemon can specifically repress the transcription of tumor suppressor gene ARF. Aims： To investigate the effect of inhibiting Pokemon by RNA interfering technique on proliferation and apoptosis of human colon cancer cell line HT-29 and its possible molecular mechanism. Methods： Interference sequence was designed according to the eDNA sequence of Pokemon for constructing the recombinant interference plasmid. HT-29 cells were transfected with the recombinant plasmid by LipofectamineTM 2000 transfection reagent. Expressions of Pokemon, pl4ARF and p53 were determined by real time RT- PCR and Western blotting, the cell cycle and apoptosis were analyzed by flow cytometry. Results： Expression levels of Pokemon mRNA （0.29 ± 0.04 ） and protein in HT-29 cells were efficiently inhibited by the siRNA targeting Pokemon gene. Meanwhile, expression levels of pl4ARF and p53 mRNA （ 3.03 ± 0.49 and 2.80±0.25 ） and protein were obviously up-regulated. Compared with HT-29 ceils transfected with siRNA not targeting Pokemon gene, Pokemon-inhibited HT-29 cells exhibited an increase in percentage of cells in G0/G1 phase （43.6% ± 2.3% vs. 34.7% ± 1.9% , P 〈 0.05 ）, accompanied by an increase in cell apoptosis （ 10.7% ± 1.9% vs. 2.7% ± 0.4%, P 〈 0.05 ）. Conclusions： Thereis a negative correlation between expression of Pokemon and expressions of pl4ARF and p53 in human colon cancer ceils. Inhibition of Pokemon may arrest cell cycle progression and induce cell apoptosis in tumor cells via up-regulating p14ARF- p53 pathway. Regulating Pokemon-p14ARF-p53 pathway might be an attractive therapeutic target for colon cancer.

关 键 词：基因 POKEMON 结肠肿瘤 细胞增殖 细胞凋亡 RNA 小分子干扰 

分 类 号：R735.35[医药卫生—肿瘤]