Correlation Between Expression of SIX1, TGF-β and VEGF-C in Primarily Cultured Human Laryngeal Squamous Cell Carcinoma Cells
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摘要:目的
探讨原代人喉鳞癌细胞中SIX1、TGF-β、VEGF-C表达相关性。
方法取新鲜人喉鳞状细胞癌组织，进行原代细胞培养，利用基因干扰技术，制备SIX1、TGF-β及SIX1+TGF-β靶向siRNA转染至人喉鳞癌细胞，转染成功后将实验细胞分为5组： A组（未转染组）、B组（转染空载体组）、C组（SIX1-siRNA组）、D组（TGFβ-siRNA组）、E组（SIX1+TGF-β-siRNA组）。Western blot及细胞爬片免疫荧光检测各组细胞SIX1、TGF-β及VEGF-C蛋白的表达。RT-PCR检测各组细胞SIX1、TGF-β及VEGF-C mRNA的表达。
结果SIX1、TGF-β、VEGF-C蛋白及其mRNA的表达在未转染组、转染空载体组中差异无统计学意义。同未转染组相比，SIX1蛋白及其mRNA的表达在SIX1-siRNA组降低的同时出现了VEGF-C表达的降低；TGF-β蛋白及其mRNA的表达在TGFβ-siRNA组降低的同时也出现了VEGF-C表达的降低，差异均有统计学意义。同SIX1-siRNA组、 TGFβ-siRNA组相比，VEGF-C蛋白及其mRNA的表达在SIX1+TGF-β-siRNA组未出现进一步降低。
结论SIX1和TGF-β能够共同作用促进VEGF-C蛋白及其 mRNA的表达，进而促进人喉鳞癌的淋巴转移。SIX1、TGF-β的变化可能成为临床抑制人喉鳞癌淋巴结转移的潜在靶点。
Abstract:ObjectiveTo investigate the relationship between sine oculis homeobox homolog 1(SIX1) , TGF-β and VEGF-C in primarily cultured human laryngeal squamous cell carcinoma cells.
MethodsWe took fresh human laryngeal squamous cell carcinoma tissues and cultured the primary cells. The technology of RNA interference was used for preparing SIX1-targeting, TGF-β-targeting and SIX1+TGF-β-targeting siRNA to transfect laryngeal squamous cell carcinoma cells. After successful transfection, the experiments were divided into five groups: Group A (untransfected), Group B(empty vector), Group C(SIX1-siRNA), Group D(TGF-β-siRNA) and Group E(SIX1+TGF-β-siRNA). The protein expression of SIX1, TGF-β and VEGF-C were determined by immunohistochemistry and Western blot. The mRNA levels of SIX1, TGF-β and VEGF-C were determined by RT-PCR.
ResultsThe protein and mRNA expression of SIX1, TGF-β and VEGF-C in empty vector group had no significant difference compared with untransfected group. In SIX1- siRNA group, the expression of SIX1 protein and mRNA were decreased, at the same time the expression of VEGF-C was decreased, with statistically significant difference compared with untransfected group. In TGF- β-siRNA group, the expression of TGF-β protein and mRNA were decreased, at the same time the expression of VEGF-C was decreased, with statistically significant difference compared with untransfected group. Compared with SIX1-siRNA and TGF-β-siRNA groups, the expression of VEGF-C protein and mRNA had no significant decrease in SIX1+TGF-β-siRNA group.
ConclusionSIX1 could promote lymph node metastasis by coordinating with TGF-β to increase the expression of VEGF-C protein and mRNA, suggesting that SIX1 and TGF-β might be potential therapeutic targets for preventing lymph node metastasis from human laryngeal squamous cell carcinoma.
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Key words:
- SIX1 /
- TGF-β /
- VEGF-C /
- Primary human laryngeal squamous cell carcinoma cells
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图 1 人喉鳞癌细胞中SIX1、TGF-β和VEGF-C蛋白表达 (×200)
Figure 1 Expressions of SIX1, TGF-β and VEGF-C proteins in human laryngeal squamous cell carcinoma cells (×200)
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图 2 人喉鳞癌细胞中SIX1、TGF-β和VEGF-C mRNA的表达
Figure 2 Expressions of SIX1, TGF-β and VEGF-C mRNA in human laryngeal squamous cell
表 1 Western blot结果显示各试验组SIX1、TGF-β和VEGF-C蛋白表达
Table 1 Expressions of SIX1, TGF-β and VEGF-C proteins in human laryngeal squamous cell carcinoma cells in each experimental groups detected by Western blot
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