Mu Ya-kui, Yu Liang, Lu Su-mei, Sa Na, Wang Hai-bo, Xu Wei
Department of Otorhinolaryngology Head and Neck Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
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Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2013 Dec;48(12):1022-7.
To investigate the effect of knockdown of EpCAM by siRNA on invasion, migration, and colony abilities in hypopharyngeal carcinoma FaDu cells.
A siRNA against EpCAM was employed to inhibit the expression of EpCAM in FaDu cells. Measurements included the Transwell assay for invasion and migration, plate colony formation assay for cell colony ability, Western blot assay for EpCAM, E-cadherin, and β-catenin expressions in total protein, cytoplasm, and cytoskeleton, respectively.
mRNA and protein expressions of EpCAM were suppressed significantly in FaDu cells transfected by EpCAM siRNA (t = 6.46, P < 0.05; t = 10.25, P < 0.05) . Transwell assay showed in transwell assay, the average invasive cells in EpCAM siRNA cells (26.33 ± 3.71) was less than that in FaDu cells (61.47 ± 6.70; t = 7.95, P < 0.05)and control cells (54.13 ± 6.51; t = 6.42, P < 0.05); the average number of migration cells in EpCAM siRNA cells (79.87 ± 8.44) was lower than that in FaDu (167.53 ± 11.49; t = 10.90, P < 0.05) cells and control cells (162.13 ± 13.45; t = 8.97, P < 0.05). In plate colony formation assay, the average colony number of EpCAM siRNA cells was (78.00 ± 5.57), which was less than that of FaDu cells(177.30 ± 16.50; t = 9.78, P < 0.05) and control cells (173.67 ± 13.50; t = 11.35, P < 0.05). Western blot assays showed, silencing of EpCAM increased the expressions of E-cadherin (t = 4.58, P = 0.01) and β-catenin (t = 3.76, P = 0.02) in cytoskeleton, and decreased the expressions of E-cadherin (t = 6.60, P < 0.05) and β-catenin (t = 8.20, P < 0.05) in cytoplasm.
The knockdown of EpCAM inhibits the invasion, migration, and colony formation abilities of FaDu cells, which is probably related to the regulation of E-cadherin and β-catenin in cytoplasm and cytoskeleton, and EpCAM may be a promising gene therapy target for hypopharyngeal carcinoma.
研究小干扰RNA（siRNA）敲低上皮细胞黏附分子（EpCAM）对下咽癌FaDu细胞侵袭、迁移及集落形成能力的影响。
采用针对EpCAM的siRNA抑制FaDu细胞中EpCAM的表达。检测指标包括：采用Transwell实验检测侵袭和迁移能力；采用平板集落形成实验检测细胞集落形成能力；采用蛋白质免疫印迹法分别检测总蛋白、细胞质及细胞骨架中EpCAM、E-钙黏蛋白和β-连环蛋白的表达。
EpCAM siRNA转染的FaDu细胞中，EpCAM的mRNA和蛋白表达均显著受到抑制（t = 6.46，P < 0.05；t = 10.25，P < 0.05）。Transwell实验显示，EpCAM siRNA组细胞的平均侵袭细胞数（26.33 ± 3.71）低于FaDu细胞组（61.47 ± 6.70；t = 7.95，P < 0.05）和对照组细胞（54.13 ± 6.51；t = 6.42，P < 0.05）；EpCAM siRNA组细胞的平均迁移细胞数（79.87 ± 8.44）低于FaDu细胞组（167.53 ± 11.49；t = 10.90，P < 0.05）和对照组细胞（162.13 ± 13.45；t = 8.97，P < 0.05）。平板集落形成实验显示，EpCAM siRNA组细胞的平均集落数为（78.00 ± 5.57），低于FaDu细胞组（177.30 ± 16.50；t = 9.78，P < 0.05）和对照组细胞（173.67 ± 13.50；t = 11.35，P < 0.05）。蛋白质免疫印迹法检测显示，敲低EpCAM可使细胞骨架中E-钙黏蛋白（t = 4.58，P = 0.01）和β-连环蛋白（t = 3.76，P = 0.02）的表达增加，使细胞质中E-钙黏蛋白（t = 6.60，P < 0.05）和β-连环蛋白（t = 8.20，P < 0.05）的表达降低。
敲低EpCAM可抑制FaDu细胞的侵袭、迁移及集落形成能力，这可能与细胞质及细胞骨架中E-钙黏蛋白和β-连环蛋白的调节有关，EpCAM可能是下咽癌有前景的基因治疗靶点。
