PI3K p55PIK调节亚单位N末端抑制胃癌细胞株MGC803生长及其机制

Inhibitory Effect of N-Terminal of p55PIK-Regulatory Subunit of Phosphoinositide-3 Kinase-on Proliferation of Gastric Cancer Cell Line MGC803 and Its Mechanism

背景与目的:p55PIK是磷脂酰肌醇3-激酶(PI3K)的调节亚单位之一。p55PIK依赖于其N末端独特的24个氨基酸结合Rb,这24个氨基酸的表达能抑制细胞周期的进程。本研究目的在于观察p55PIKN末端24个氨基酸的表达对胃癌细胞增殖和生长的影响,并初步探讨其可能的作用机制。方法:通过Lipofectamine介导,用pEGFP-N24表达质粒转染MGC803细胞,用Westernblot鉴定融合蛋白GFP-N24在MGC803细胞中的瞬时表达;MTT法检测转染细胞的生长情况;克隆形成实验观察N24p55PIK过表达对细胞克隆形成能力的影响;动物实验观察表达N24p55PIK的细胞在裸鼠体内的成瘤能力;Westernblot法分析N24p55PIK表达对细胞周期蛋白CyclinD1表达的影响。结果:N24p55PIK在MGC803细胞中能够有效表达,但其表达量明显低于对照组。与对照组细胞相比,N24p55PIK的表达使细胞生长速度减慢,细胞倍增时间明显延长;表达N24p55PIK的细胞在克隆形成的数量上与对照组相比差异无显著性,但在克隆体积上明显小于对照组。表达N24p55PIK的MGC803细胞在裸鼠中的成瘤能力有所下降,其所形成肿瘤的重量和体积分别为(0.398±0.244)g和(408±268)mm3,而空载体对照组分别为(0.763±0.193)g和(829±271)mm3,两组相比差异有显著性(P<0.05)。N24p55PIK在MGC803细胞中的表达抑制CyclinD1的表达。结论:p55PIK调节亚单位N末端24个氨基酸的表达在体外和体内均能抑制肿瘤细胞的生长;减少CyclinD1的表达可能是其发挥作用的主要机制;来源于PI3K调节亚单位p55PIK的N24肽在肿瘤的治疗上可能具有潜在的应用前景。

BACKGROUND ＆ OBJECTIVE： p55PIK is one of the regulatory subunits of phosphoinositide-3 kinase （PI3K）. The unique 24 amino acids at N-terminal of p55PIK can bind Rb, and their ectopic expression may inhibit cell cycle progression. This study was to observe the effects of ectopic expression of the 24 amino acids at N-terminal of p55PIK （N24p55PIK） on cell proliferation and tumor growth of gastric cancer, and explore possible mechanism. METHODS： Plasmid pEGFPN24 was transfected into gastric cancer cell line MGC803 （MGC803/GFP-N24）; pEGFPC1 was transfected into MGC803 cells as control （MGC803/ pEGFPC1）. Transient expression of GFP-N24 fusion protein was confirmed by Western blot. The growth of cell clones was determined by M-IF assay. Effect of N24p55PIK overexpression on cell clonogenic ability was detected by colony formation assay. Tumorigenic capacity of MGC803/GFR-N24 cells was tested by tumorigenicity assay in nude mice. Influence of N24p55PIK on the expression of cell cycle protein Cyclin D1 was analyzed by Western blot. RESULTS： N24p55PIK was efficiently expressed in MGC803 cells, but the level of GFP-N24 fusion protein in MGC803/GFP-N24 cells was much lower than that of GFP in MGC803/pEGFPC1 cells. Compared with MGC803/ pEGFPC1 cells, the growth of MGC803/GFP-N24 cells was suppressed and the cell doubling time was prolonged. The volume of MGC803/GFP-N24 cell colonies was smaller than that of MGC803/pEGFPC1 cell colonies. The tumorigenic capacity of MGC803 cells was decreased after transfection of pEGFPN24 in nude mice. The tumor weight and volume were （0.398±0.244） g and （408±268） mm3 in MGC803/pEGFPN24 group, and were （0.763± 0.193） g and （829±271） mm3 in MGC803/pEGFPC1 group （P〈0.05）. The expression of Cyclin D1 was down-regulated in MGC803/GFP-N24 cells. CONCLUSIONS： Ectopic expression of N24p55PIK might inhibit tumor cell growth both in vitro and in vivo through decreasing the expression of Cyclin D1. The N24 peptide, derived from PI3K regulatory subunit p55PIK, may be a potential drug in antitumor treatment.