Id3过表达对不同肿瘤细胞中β-catenin的调节

发布时间：2018-07-11 11:15

本文选题：分化抑制因子 + β-catenin　；参考：《医学研究生学报》2017年05期

【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(P0.05);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(P0.05)。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(P0.05),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(P0.05)。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,P0.01)。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,P0.01);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(P0.05)。Western blot检测结果显示,与对照组比较,Id3过表达后可明显下调肠癌细胞SW-480中β-catenin的表达水平,而肺癌细胞A549和A549/DDP中β-catenin的表达水平则无明显变化。结论 Id3和β-catenin在不同的肿瘤细胞中有不同的表达水平,提示β-catenin在肠癌细胞中的异常高表达是引起肠癌的重要因素之一;外源性转染Id3基因抑制肠癌SW-480细胞中β-catenin的表达,有望为肠癌靶基因治疗提供新思路。
[Abstract]:Objective differentiation inhibitory factor 3 (Id3) is involved in tumorigenesis, cell proliferation and apoptosis, and 尾 -catenin is the key to tumorigenesis. To investigate the expression of Id3 and 尾 -catenin in different tumor cells and the regulatory effect of Id3 on 尾 -catenin. Methods the total RNAs of tumor cells were extracted by Trizol method. The relative expressions of Id3 and 尾 -catenin in tumor cells were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The eukaryotic expression vector pEGFP / Id3 containing human Id3 gene was transfected into SW-480 cells by non-liposome transfection technique. The expression of EGFP-Id3 fusion protein was observed by fluorescence microscope and the expression level of Id3 and 尾 -catenin mRNA in transfected cells was analyzed by qRT-PCR. The expression level of Id3 and 尾 -catenin protein in transfected cells was analyzed by Western blot. Results Id3 expression was the lowest in SW-480 and HT-29 cells. The expression of 尾 -catenin in CNE5-8F cells of nasopharyngeal carcinoma was significantly higher than that of other cancer cells (P0.05) .Id3 expression was the lowest in colorectal cancer SW-480, and the highest 尾 -catenin expression was found in cancer cells with lower expression of AGS than other cancer cells (P0.05), and the expression of 尾 -catenin was significantly higher than that of other cancer cells (P0.05), and the expression of 尾 -catenin was significantly higher than that of other cancer cells (P0.05). The expression of 尾 -catenin in HT-29 cells was the second. The expression of 尾 -catenin in other tumor cells such as H446A549SPC-A-1A549 / DDPnSK-MES-1 cells was low, while the expression of 尾 -catenin in CNE5-8F cells with high Id3 expression was very low or almost non-expressed, and there was significant difference compared with other groups (P0.05). Fluorescence microscopy showed that the cells transfected with Id3 / p EGFP decreased in volume, the cell membrane shrank, and the diopter disappeared. However, after transfection with empty vector pEGFP, most of the cells did not show the above changes. Compared with the control group, the expression of Id3 mRNA in A549 / DDIP-SW-480 cells was significantly higher than that in the control group (1.24 卤0.12 vs 193.12 卤2.800.09 卤0.11 vs 188.30 卤2.600.92 卤0.29 vs 19.08 卤0.59P0.01), and the expression of Id3 mRNA was significantly higher than that in the control group (1.24 卤0.12 vs 193.12 卤2.80,0.09 卤0.11 vs 188.30 卤2.600.92 卤0.29 vs 19.08 卤0.59P0.01). Compared with the control group, 尾 -catenin mRNA was significantly down-regulated in Id3 overexpressed SW-480 cells (0.98 卤0.05 vs 0.32 卤0.03P0.01), but there was no significant difference in 尾 -catenin expression in A549 and A549-DDP cells after transfection (P0.05). Compared with the control group, the expression of 尾 -catenin in SW-480 cells was significantly down-regulated, but the expression level of 尾 -catenin in A549 and A549 / DDP cells was not significantly changed. Conclusion there are different expression levels of Id3 and 尾 -catenin in different tumor cells, suggesting that the abnormal overexpression of 尾 -catenin in intestinal cancer cells is one of the important factors leading to colorectal cancer, and exogenous transfection of Id3 gene inhibits the expression of 尾 -catenin in SW-480 cells. It is expected to provide a new idea for target gene therapy of colorectal cancer.
【作者单位】：南方医科大学金陵医院(南京军区南京总医院)中心实验科;
【基金】：国家自然科学基金(81171652)
【分类号】：R730.2

【相似文献】