中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2013年
12期
1022-1027
,共6页
牟亚魁%于亮%逯素梅%洒娜%王海波%徐伟
牟亞魁%于亮%逯素梅%灑娜%王海波%徐偉
모아괴%우량%록소매%쇄나%왕해파%서위
下咽肿瘤%RNA,小分子干扰%细胞黏附分子%抗原,肿瘤%细胞系,肿瘤
下嚥腫瘤%RNA,小分子榦擾%細胞黏附分子%抗原,腫瘤%細胞繫,腫瘤
하인종류%RNA,소분자간우%세포점부분자%항원,종류%세포계,종류
Hypopharyngeal neoplasms%RNA,small interfering%Cell adhesion molecules%Antigens,neoplasm%Cell line,tumor
目的 研究小干扰RNA(siRNA)干扰上皮细胞黏附因子(epithelial cell adhesion molecule,EpCAM)表达对下咽癌FaDu细胞体外侵袭、迁移、克隆形成能力的影响,并分析其可能机制.方法 利用siRNA技术干扰FaDu细胞中EpCAM基因的表达;采用Transwell侵袭、迁移实验和平板克隆实验,分别检测干扰EpCAM表达对FaDu细胞侵袭、迁移及克隆形成能力的影响;借助Western blot检测干扰EpCAM表达对E-cadherin和β-catenin在细胞质及细胞骨架上表达的影响.结果 FaDu细胞转染EpCAM siRNA后,EpCAM在mRNA和蛋白水平表达较阴性对照组均显著下调(t =6.46,P<0.05;t=10.25,P<0.05).侵袭实验显示:EpCAM siRNA组每个视野下平均细胞数目为(26.33 ±3.71)个,较FaDu组(61.47 ±6.70)个及阴性对照组(54.13 ±6.51)个下降明显,差异有统计学意义(t =7.95,t =6.42,P<0.05).迁移实验显示:EpCAM siRNA组每个视野下平均细胞数目为(79.87 ±8.44)个,低于FaDu组(167.53 ±11.49)个和阴性对照组(162.13±13.45)个,与阴性对照组相比差异有统计学意义(t =10.90,t=8.97,P<0.05).平板克隆实验中,EpCAM siRNA组平均克隆形成数目为(78.00 ±5.57)个,低于FaDu组(177.30±16.50)个和阴性对照组(173.67±13.50)个,差异具有统计学意义(=9.78,=11.35,P<0.05).Westem blot显示,EpCAM siRNA组E-cadherin和β-catenin在细胞骨架上表达水平显著升高,与阴性对照组相比差异均具有统计学意义(=4.58,P<0.05;t =3.76,P<0.05);而在细胞质中表达则显著降低,与阴性对照组相比差异有统计学意义(t =6.60,P<0.05;t=8.20,P<0.05);在总蛋白水平,E-cadherin和β-catenin表达无明显变化.结论 干扰FaDu细胞中EpCAM基因的表达,抑制细胞的侵袭、迁移和克隆形成能力,其机制可能与调节E-cadherin和β-catenin在细胞骨架和细胞质中的表达有关,EpCAM基因有可能成为下咽癌基因治疗的新靶点.
目的 研究小榦擾RNA(siRNA)榦擾上皮細胞黏附因子(epithelial cell adhesion molecule,EpCAM)錶達對下嚥癌FaDu細胞體外侵襲、遷移、剋隆形成能力的影響,併分析其可能機製.方法 利用siRNA技術榦擾FaDu細胞中EpCAM基因的錶達;採用Transwell侵襲、遷移實驗和平闆剋隆實驗,分彆檢測榦擾EpCAM錶達對FaDu細胞侵襲、遷移及剋隆形成能力的影響;藉助Western blot檢測榦擾EpCAM錶達對E-cadherin和β-catenin在細胞質及細胞骨架上錶達的影響.結果 FaDu細胞轉染EpCAM siRNA後,EpCAM在mRNA和蛋白水平錶達較陰性對照組均顯著下調(t =6.46,P<0.05;t=10.25,P<0.05).侵襲實驗顯示:EpCAM siRNA組每箇視野下平均細胞數目為(26.33 ±3.71)箇,較FaDu組(61.47 ±6.70)箇及陰性對照組(54.13 ±6.51)箇下降明顯,差異有統計學意義(t =7.95,t =6.42,P<0.05).遷移實驗顯示:EpCAM siRNA組每箇視野下平均細胞數目為(79.87 ±8.44)箇,低于FaDu組(167.53 ±11.49)箇和陰性對照組(162.13±13.45)箇,與陰性對照組相比差異有統計學意義(t =10.90,t=8.97,P<0.05).平闆剋隆實驗中,EpCAM siRNA組平均剋隆形成數目為(78.00 ±5.57)箇,低于FaDu組(177.30±16.50)箇和陰性對照組(173.67±13.50)箇,差異具有統計學意義(=9.78,=11.35,P<0.05).Westem blot顯示,EpCAM siRNA組E-cadherin和β-catenin在細胞骨架上錶達水平顯著升高,與陰性對照組相比差異均具有統計學意義(=4.58,P<0.05;t =3.76,P<0.05);而在細胞質中錶達則顯著降低,與陰性對照組相比差異有統計學意義(t =6.60,P<0.05;t=8.20,P<0.05);在總蛋白水平,E-cadherin和β-catenin錶達無明顯變化.結論 榦擾FaDu細胞中EpCAM基因的錶達,抑製細胞的侵襲、遷移和剋隆形成能力,其機製可能與調節E-cadherin和β-catenin在細胞骨架和細胞質中的錶達有關,EpCAM基因有可能成為下嚥癌基因治療的新靶點.
목적 연구소간우RNA(siRNA)간우상피세포점부인자(epithelial cell adhesion molecule,EpCAM)표체대하인암FaDu세포체외침습、천이、극륭형성능력적영향,병분석기가능궤제.방법 이용siRNA기술간우FaDu세포중EpCAM기인적표체;채용Transwell침습、천이실험화평판극륭실험,분별검측간우EpCAM표체대FaDu세포침습、천이급극륭형성능력적영향;차조Western blot검측간우EpCAM표체대E-cadherin화β-catenin재세포질급세포골가상표체적영향.결과 FaDu세포전염EpCAM siRNA후,EpCAM재mRNA화단백수평표체교음성대조조균현저하조(t =6.46,P<0.05;t=10.25,P<0.05).침습실험현시:EpCAM siRNA조매개시야하평균세포수목위(26.33 ±3.71)개,교FaDu조(61.47 ±6.70)개급음성대조조(54.13 ±6.51)개하강명현,차이유통계학의의(t =7.95,t =6.42,P<0.05).천이실험현시:EpCAM siRNA조매개시야하평균세포수목위(79.87 ±8.44)개,저우FaDu조(167.53 ±11.49)개화음성대조조(162.13±13.45)개,여음성대조조상비차이유통계학의의(t =10.90,t=8.97,P<0.05).평판극륭실험중,EpCAM siRNA조평균극륭형성수목위(78.00 ±5.57)개,저우FaDu조(177.30±16.50)개화음성대조조(173.67±13.50)개,차이구유통계학의의(=9.78,=11.35,P<0.05).Westem blot현시,EpCAM siRNA조E-cadherin화β-catenin재세포골가상표체수평현저승고,여음성대조조상비차이균구유통계학의의(=4.58,P<0.05;t =3.76,P<0.05);이재세포질중표체칙현저강저,여음성대조조상비차이유통계학의의(t =6.60,P<0.05;t=8.20,P<0.05);재총단백수평,E-cadherin화β-catenin표체무명현변화.결론 간우FaDu세포중EpCAM기인적표체,억제세포적침습、천이화극륭형성능력,기궤제가능여조절E-cadherin화β-catenin재세포골가화세포질중적표체유관,EpCAM기인유가능성위하인암기인치료적신파점.
Objective To investigate the effect of knockdown of EpCAM by siRNA on invasion,migration,and colony abilities in hypopharyngeal carcinoma FaDu cells.Methods 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.Results 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.Conclusions 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.