RNA干扰介导的PIK3CA基因沉默抑制Hep-2细胞增殖和侵袭力

A study on cells proliferation and invasiveness of Hep-2 inhibited by RNA interference mediated PIK3CA gene silencing

目的观察RNA干扰介导PIK3CA基因表达沉默对喉鳞状细胞癌(简称喉癌)细胞增殖和侵袭力的影响,探讨PIK3CA基因作为喉癌基因治疗靶标的可行性。方法构建PIK3CA-shRNA的慢病毒表达载体,在脂质体介导下转染人喉癌细胞Hep-2。采用real-time RT-PCR和Western-blot检测PIK3CA基因的表达,MTT法、细胞克隆形成实验、细胞生长曲线检测细胞生长增殖,Boyden小室模型检测细胞的体外侵袭力。结果表达PIK3CA-shRNA的慢病毒载体构建成功;与对照组相比,实验组细胞PIK3CA mRNA和蛋白表达明显下调,分别达75%和70%(P<0.05);细胞增殖和侵袭力均受到明显抑制,差异显著(P<0.05)。结论靶向PIK3CA基因的RNA干扰可抑制喉癌细胞的增殖和侵袭力,PIK3CA有望成为喉癌基因治疗的新的候选基因。

OBJECTIVE To observe the effects of RNA interference mediated PIK3CA gene silencing on the proliferation and invasiveness of laryngeal squamous cell carcinoma （LSCC） cells, and investigate the feasibility of PIK3CA gene as a potential therapeutic target in the treatment of LSCC. METHODS The lentiviral vector system expressing short hairpin RNAtargeting PIK3CA gene （PIK3C-shRNA） was constructed and transfected subsequently into Hep-2 cells mediated by liposome in vitro. The expression of PIK3CA gene was detected by real-time RT-PCR and Western blot respectively. The proliferation of Hep-2 cells was measured by MTT, colony formation, and cell growth curve. The invasive power was determined by Boyden chamber model in vitro. RESULTS The lentiviral vector system expressing short hairpin PIK3CA-shRNA was constructed successfully. Compared with the control groups, the mRNA and protein expression of PIK3CA were significantly down-regulated （75% and 70% respectively） in the experimental group （P〈0.01） . The cell proliferation and invasive power were significantly inhibited in vitro （P〈0.05） . CONCLUSION The results suggest that shRNA mediated PIK3CA gene silencing can inhibit the proliferation and invasiveness of Hep-2 cells in vitro. PIK3CA has the possibility to be a new candidate gene for gene therapy of LSCC.