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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。
Purpose Differentiation inhibitor 3 (Id3) is involved in the process of tumorigenesis, cell proliferation and apoptosis; β-catenin is the key to tumorigenesis. In this paper, the expression of Id3 and β-catenin in different tumor cells and Id3 regulation of β-catenin. Methods Trizol method was used to extract the total RNA of tumor cells. Real-time quantitative PCR (qRT-PCR) was used to analyze the relative expression of Id3 and β-catenin in tumor cells. Non-liposome transfection was used to detect the expression of Id3 The eukaryotic expression vector pEGFP / Id3 was transfected into SW-480, human lung adenocarcinoma A549 and human lung adenocarcinoma A549 / DDP cells respectively. The expression of EGFP-Id3 fusion protein was observed by fluorescence microscopy The expression of Id3 and β-catenin mRNA in transfected cells were detected by qRT-PCR. The expression of Id3 and β-catenin in transfected cells were detected by Western blot. Results The expression of Id3 was the lowest in colorectal cancer SW-480 and HT-29 cells, which was significantly lower than that in A549 and other tumor cells (P <0.05). The expression of Id3 in nasopharyngeal carcinoma CNE and 5-8F cells was significantly higher than other Tumor cell group (P <0.05). Β-catenin in SW480 of SW480 with the lowest expression of Id3 was the highest (P <0.05), the expression of β-catenin was lower in AGS cells with lower Id3 expression and HT-29 cells with colon cancer, The expression of β-catenin in other tumor cells such as H446, A549, SPC-A-1, A549 / DDP and SK-MES-1 cells was low, while in CNE and 5-8F, The content of -catenin was relatively low or almost not expressed, and the difference was statistically significant compared with other groups (P <0.05). Fluorescence microscopy showed that the cells transfected with Id3 / p EGFP were reduced in size, the cell membrane was shrunk and the refraction disappeared. However, no changes were observed in most of the cells transfected with empty vector p EGFP. Compared with the control group, Id3 mRNA expression of Id3 / p EGFP group was significantly increased after transfected 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, 0.92 ± 0.29 vs 19.08 ± 0.59, P <0.01). Compared with the control group, the expression of β-catenin mRNA was significantly down-regulated in SW380 overexpression cells (0.98 ± 0.05 vs 0.32 ± 0.03, P <0.01), while in A549 and A549 / DDP cells, The difference of β-catenin expression was not statistically significant (P> 0.05). Western blot results showed that, compared with the control group, Id3 overexpression significantly down-regulated the expression of β-catenin in SW-480 colon cancer cells, while the expression of β-catenin in lung cancer A549 and A549 / DDP groups was not significant Variety. Conclusions Id3 and β-catenin have different expression levels in different tumor cells, suggesting that abnormally high expression of β-catenin in colorectal cancer cells is one of the important factors causing colorectal cancer. Exogenous transfection of Id3 gene inhibits intestinal The expression of β-catenin in SW-480 cells is expected to provide new ideas for the target gene therapy of colorectal cancer.