T细胞因子4显性负调节基因ΔNTCF4对肾癌细胞GRC-Ⅰ生物学性状的影响

Effects of dominant-negative truncation mutant ΔNTCF4 on biological characteristics of renal cancer cell line GRC-I by down-regulation Wnt signaling pathway target genes

目的观察T细胞因子4(TCF4)显性负调节基因ΔNTCF4对肾癌细胞GRC-1生长、增殖等生物学行为的影响。方法将缺失N末端的TCF4显性负调节基因真核表达质粒pCDNA3-ΔNTCF4和pCDNA3空载体分别转染肾癌细胞GRC-Ⅰ,建立稳定表达ΔNTCF4基因的肾癌细胞系GRC-Ⅰ/ΔNTCF4和空载体细胞系GRC-Ⅰ/Mock,光镜下观察细胞生长状态,绘制生长曲线,MTT法检测细胞增殖活性,免疫细胞化学和Western blot检测Wnt信号通路下游靶基因C-Myc、Cox-2蛋白表达水平。结果稳定表达ΔNTCF4基因的GRC-Ⅰ/ΔNTCF4细胞形态由圆形变为长条状,细胞生长速度变慢,核分裂相减少,出现向正常肾小管细胞逆转的趋势；GRC-Ⅰ/ΔNTCF4细胞增殖活性受到明显抑制,与GRC-Ⅰ对照细胞相比抑制率达11．2%～35．5%(P＜0．05),而GRC-Ⅰ/Mock与对照细胞相比差异无统计学意义；表达显性负调节基因的肾癌细胞系GRC-Ⅰ/ΔNTCF4中Cox-2蛋白表达水平显著降低。结论转染T细胞因子4显性负调节基因ΔNTCF4能部分抑制肾癌细胞GRC-Ⅰ生长,降低Wnt通路靶基因C-Myc、Cox-2表达,为通过阻断Wnt信号通路对肾癌进行细胞信号治疗提供了实验依据。

Objective To investigate the effects of dominant-negative truncation mutant △NTCF4, lacking the N-terminal form of TCF4 gene, on biological characteristics of renal cancer cell line GRC-Ⅰ and explore the molecular mechanisms. Methods GRC-Ⅰ cell was transfeeted with pCDNA3-△NTCF4 enkaryotic expression plasmid, pCDNA3 empty vector to construct the stable cell line GRC-Ⅰ/△NTCF4 and GRC-Ⅰ/ Mock respectively. The morphological changes of stable cells were observed and the cells growth curve was detected through light microscope. The cellular proliferation activities were determined using the MTT assay. The protein expression of Wnt pathway downstream target gene C-Myc and Cox-2 was evaluated by inlmunocytochemical method and Western Blot analysis. Results After the dominant-negative △NTCF4 gene was permanently expressed, the GRC-Ⅰ/△NTCF4 stable cells morphologically showed that appearance changed from circular to long-spindle shape, growth rate decreased with less karyosehisis found, malignant phenotypes reversed to normal renal tabular cells. MTT assay revealed that the proliferation activities of GRC-Ⅰ/△NTCF4 cells were inhibited by 11.2% -35.5% compared with GRC-Ⅰ cells （P 〈 0.05 ） , while the GRC- Ⅰ/Mock cells have no difference with the control cells. Immunocytochemical analysis and Western Blot showed that the C-Mye and Cox-2 protein expression level of GRC-Ⅰ/△NTCF4 cells were significantly suppressed in comparison with that of GRC-Ⅰ/Mock and GRC-Ⅰ cells. Conclusions The dominant-negative truncation mutant △NTCF4 could partially inhibit the growth of renal cancer cells and down-regulate the prorein expression of Wnt pathway target gene C-Myc and Cox-2. These findings provide a experimental foundation for applying cell signal therapy to renal cell cancer by blocking the Wnt signaling pathway.