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目的探讨钙周期素结合蛋白(CacyBP)对胃癌细胞增殖的影响及可能的机制。方法构建CacyBP小干扰RNA(siRNA)表达载体,脂质体法转染至人胃癌细胞系SGC7901,四甲基偶氮唑盐(MTT)比色法观察细胞生长,平板克隆实验及裸鼠成瘤实验检测转染细胞的体外和体内的成瘤性,通过Western印迹及半定量逆转录-聚合酶链反应(RT-PCR)检测转染细胞中一些相关分子的表达。结果成功构建了CacyBP的siRNA表达载体,转染SGC7901细胞后显著抑制了内源性CacyBP的表达。CacyBP表达下调的胃癌转染细胞生长加快;SGC/CacyBP-siRNA1细胞(0·35±0·03)和SGC/CacyBP-siRNA2细胞(0·43±0·05)克隆形成率显著高于对照细胞(0·18±0·03)(P<0·01),体外成瘤能力增强;SGC/CacyBP-siRNA荷瘤裸鼠形成的瘤体明显大于对照组小鼠(P<0·001),并使荷瘤裸鼠的生存期显著缩短(P<0·01)。转染细胞中COX-2和细胞周期蛋白D1的mRNA和蛋白均显著增加,β-连环蛋白、rac1和热休克蛋白70的蛋白水平升高,而mRNA无明显变化。结论CacyBP表达下调能够促进胃癌细胞的恶性增殖。
Objective To investigate the effect of CacyBP binding protein (CacyBP) on the proliferation of gastric cancer cells and its possible mechanism. Methods CacyBP small interfering RNA (siRNA) expression vector was constructed and transfected into human gastric cancer cell line SGC7901 by lipofectamine. MTT assay was used to observe cell growth, plate cloning and tumorigenesis in nude mice The in vitro and in vivo tumorigenicity of the transfected cells was examined experimentally, and the expression of some related molecules in transfected cells was detected by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Results The CacyBP siRNA expression vector was successfully constructed. The transfection of SGC7901 cells significantly inhibited the expression of CacyBP. The rate of colony-forming of SGC / CacyBP-siRNA1 cells (0 · 35 ± 0 · 03) and SGC / CacyBP-siRNA2 cells (0 · 43 ± 0 · 05) was significantly higher than that of control cells (0 · 18 ± 0 · 03) (P <0.01), and the ability of tumorigenesis was enhanced in vitro. The tumors formed by SGC / CacyBP-siRNA tumor-bearing nude mice were significantly larger than those of the control mice And significantly shortened the survival of tumor-bearing nude mice (P <0.01). The mRNA and protein expressions of COX-2 and cyclin D1 were significantly increased in transfected cells, and the protein levels of β-catenin, rac1 and HSP70 increased, while the mRNA did not change significantly. Conclusion The down-regulation of CacyBP can promote the malignant proliferation of gastric cancer cells.