RNA干扰沉默Aurora-A基因抑制喉癌Hep-2细胞生长的实验研究

Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth

目的 研究RNA干扰沉默Aurora-A基因后对喉癌Hep-2细胞体外体内生长的抑制作用.方法 构建靶向Aurora-A的小干扰RNA（siRNA）表达质粒并转染喉鳞癌Hep-2细胞株,采用CCK-8实验、Transwell实验、软琼脂克隆形成实验和裸鼠体内接种观察Aurora-A沉默后Hep-2细胞的增殖、侵袭和克隆形成能力以及体内肿瘤生长情况,Western blot和免疫组化检测参与细胞黏附侵袭的重要因子黏着斑激酶和基质金属蛋白酶-2的表达情况.结果 Hep-2细胞转染Aurora-A siRNA 后,siRNA-3实验组的Aurora-A蛋白的表达下降了52％,CCK-8实验中siRNA-3组细胞的平均（x±s）吸光度值为（3.268±0.106）,低于Hep-2细胞组的（3.722 ±0.152）,差异有统计学意义（F=17.634,P＜0.01）；Transwell实验显示,siRNA-3组每个视野的平均侵袭细胞数目为（110.0±18.0）个,较Hep-2组的（236.0 ±26.0）个减少,两组差异有统计学意义（F=26.462,P＜0.01）；克隆形成实验中,siRNA-3组平均集落数目（31.0±6.6）个,低于Hep-2组平均细胞集落数目（104.0±14.0）个（F=75.321,P＜0.001）.接种后,体内移植瘤生长受到抑制,siRNA-3组的肿瘤平均体积为（127.77±174.83） mm3,低于Hep-2组的肿瘤平均体积（837.26±101.80） mm3（F=28.187,P＜0.001）.在体外和体内黏着斑激酶和基质金属蛋白酶-2的表达也下降.结论 抑制Aurora-A基因后,通过降低黏着斑激酶和基质金属蛋白酶-2的表达,抑制Hep-2细胞的生长,减弱肿瘤细胞的恶性侵袭性,Aurora-A可以成为喉鳞癌治疗良好的干预靶点.

Objective To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo.Methods A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation,Transwell assay for invasion,colony formation assay for cell anchorage-independent growth.Western blot and immunohistochemistry assay for protein expression.Tumorigenicity was observed in vivo.Results In Hep-2 cells transfected by Aurora-A siRNA （ designated as siRNA-3）,protein expression of Aurora-A was suppressed by 52%.In CCK-8 assay,absorbance value of siRNA-3 cells （3.268 ±0.106,x ±s） was lower than that of Hep-2 cells （3.722 ±0.152,F =17.634,P 〈 0.001 ）.In Transwell assay,the average invasive cells per field in siRNA-3 cells （ 110.0 ± 18.0） was less than that in Hep-2 cells （236.0 ± 26.0,F =26.462,P 〈 0.01 ）.In colony formation assay,the average colony number of siRNA-3 cells （31.0 ±6.6） was lower than that of Hep-2 cells （ 104.0 ± 14.0）.The average tumor size in siRNA-3 group was （ 127.77 ± 174.83 ） mm3,which was less than Hep-2 cell group （837.26 ± 101.80） mm3 （ F =28.187,P 〈 0.001 ）.Silencing of Aurora-A decreased the expression of focal adhesion kinase（FAK） and matrix metalloproteinase-2 （MMP-2）,key regulators in cell adhesion and invasion.Conclusions The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo,which may be a promising therapeutic strategy for LSCC.