摘要
目的分化抑制因子3(Id3)参与肿瘤发生、细胞增殖和凋亡等过程;β-catenin是导致肿瘤发生的关键。文中探讨Id3与β-catenin在不同肿瘤细胞中的表达及Id3对β-catenin的调控作用。方法采用Trizol法提取各肿瘤细胞总RNA,运用实时荧光定量PCR技术(qRT-PCR)分析肿瘤细胞中Id3和β-catenin的相对表达量;用非脂质体转染技术将含人Id3基因的真核表达载体p EGFP/Id3分别导入SW-480、人肺腺癌细胞A549和人肺腺癌细胞耐顺铂株A549/DDP 3种细胞,荧光显微镜观察细胞内EGFP-Id3融合蛋白的表达情况;qRT-PCR技术分析转染后细胞内Id3及β-catenin mRNA的表达水平;Western blot分析转染后细胞内Id3及β-catenin蛋白的表达水平。结果 Id3在肠癌SW-480及HT-29细胞中表达量最低,明显低于A549及其他肿瘤细胞(P<0.05);在鼻咽癌CNE、5-8F细胞中的表达量显著高于其他肿瘤细胞组(P<0.05)。Id3表达量最低的肠癌SW-480中β-catenin与其他组细胞相比含量最高(P<0.05),Id3表达较低的胃癌AGS细胞及肠癌HT-29细胞β-catenin表达次之,其他肿瘤细胞如H446、A549、SPC-A-1、A549/DDP、SK-MES-1细胞中β-catenin均呈低表达,而Id3表达量较高的CNE、5-8F等肿瘤细胞中β-catenin的含量相对极低或几乎不表达,且与其他组细胞相比差异有统计学意义(P<0.05)。荧光显微镜观察发现,转染Id3/p EGFP的细胞体积缩小,细胞膜皱缩,折光度消失,而转染空载体p EGFP后,大部分细胞未见上述变化。与对照组相比,Id3/p EGFP组A549、A549/DDI、SW-480细胞转染后Id3 mRNA表达水平均有显著增高(1.24±0.12 vs 193.12±2.80,1.09±0.11 vs 188.30±2.60,0.92±0.29 vs 19.08±0.59,P<0.01)。与对照组比较,β-catenin mRNA在Id3过表达的肠癌SW-480细胞中表达明显下调(0.98±0.05 vs 0.32±0.03,P<0.01);而在A549和A549/DDP细胞中,Id3转染后β-catenin表达水平差异无统计学意义(P>0.05)。Western blot检测结果显示,与对照组比较,Id3过表达后可明显下调�
Objective The inhibitor of differentiation 3（ Id3） is an important transcriptional regulation factor,which participates in tumorigenesis,cell proliferation,and cell apoptosis. β-catenin,as a central molecule of the Wnt signaling pathway,is critical for tumor development. This study aimed to evaluate the expressions of these two molecules and the regulatory effect of Id3 on β-catenin in different tumor cells. Methods Total RNA was extracted using the Trizol Reagent. The relative mRNA expression levels of Id3 andβ-catenin in tumor cells were detected by quantitative real-time PCR（ qRT-PCR）. The recombinant eukaryotic expression vector p EGFP/Id3 with the human Id3 gene was transfected into A549,A549/DDP and SW-480 cells using the non-liposome-mediated method.The protein expressions of Id3 and β-catenin were determined by Western blot. Results The expression of Id3 was significantly lower in the colorectal cancer cell lines SW-480 and HT-29 than in A549 and other tumor cells（ P〈 0.05）,but remarkably higher in nasopharyngeal carcinoma CNE and 5-8F cells than in other tumor cells（ P 〈 0. 05）. The expression of β-catenin was the highest in SW-480 in comparison withother malignant tumor cells（ P〈0.05）,and the second highest was in gastric cancer AGS and colorectal cancer HT-29 cell lines,but low in H446,A549,SPC-A-1,A549/DDP,and SK-MES-1 cell lines and extremely low or almost absent in CNE and 5-8F cells（ P〈0.05）.After transfected with p EGFP/Id3,the cells showed a decreased volume,wrinkled membrane and absent refraction under the fluorescence microscope,which,however,were not observed in most of the cells transfected with the empty vector p EGFP. Compared with the control,the Id3/p EGFP group showed remarkably increased expressions of Id3 mRNA in the A549,A549/DDI,and SW-480 cells（ 1.24±0.12 vs 193.12±2.80,1.09±0.11 vs 188.30± 2.60,and 0.92± 0.29 vs 19.08± 0.59,P〈 0.01）,and the expression of β-catenin was significantly down-regulated in the transfected SW-480 cells with
出处
《医学研究生学报》
CAS
北大核心
2017年第5期464-469,共6页
Journal of Medical Postgraduates
基金
国家自然科学基金(81171652)
