钙周期素结合蛋白对胃癌细胞生长增殖的影响

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。

The effect of calcyclin binding protein on gastric cancer cell proliferation

OBJECTIVE: To study the effect of calcyclin binding protein (CacyBP) on the proliferation of gastric cancer cells. METHODS: CacyBP siRNA expression vector was constructed and transfected into the gastric cancer cells of the line SGC7901 (SGC/CacyBP-siRNA cells). Blank vector mU6 was transfected too as control group (SGC/mU6 cells). Western blotting and semi-quantitative RT-PCR were used to detect the protein expression and mRNA expression of CacyBP in the transfected cells. Immunofluorescence staining was used to detect the intensity of green fluorescence. The cell growth was determined by MTT method. Western blotting and semi-quantitative RT-PCR were used to detect the protein expression and mRNA expression of beta-catenin, cyclooxygenase-2 (COX-2), cyclin D1, rac1, and heat shock protein (HSP) 70. The protein level of beta-catenin in the nuclei of the transfected cells was detected. Twenty-fife nude mice were randomly divided into 5 groups to be inoculated with the SGC7901 cells stably transfected with CacyBP siRNA expression vector and the size of tumor was observed 1, 2, 3, 4, and 5 weeks after the inoculation respectively. The blank vector mU6 was inoculated as control group. RESULTS: The mRNA expression and protein expression of endogenous CacyBP in the SGC/CacyBP-siRNA cells were both markedly lower than those of the SGC/mU6 cells. Immunofluorescence staining showed weaker green fluorescence in the SGC/CacyBP-siRNA cells than the SGC/mU6 cells. MTT method showed that the growth of the SGC/CacyBP-siRNA cells was significantly faster than that of the SGC/mU6 cells (P < 0.01). Western blotting showed remarkable up-regulation of the protein expression of beta-catenin, COX-2, cyclin D1, rac1, and HSP 70 in the SGC/CacyBP-siRNA cells; and semi-quantitative RT-PCR showed remarkable up-regulation of the mRNA expression of COX-2 and cyclin D1 in the SGC/CacyBP-siRNA cells, however, the mRNA expression of HPS70, rac1, and beta-catenin showed no significant differences between these 2 groups. The size of tumor of the mice inoculated with SGC/CacyBP-siRNA cells was significantly larger than that of the mice inoculate with SGC/mU6 cells (P < 0.01). The survival times of the nude mice inoculated with SGC/CacyBP-siRNA1 and SGC/CacyBP-siRNA1 cells respectively were 60 d +/- 8 d and 50 d +/- 4 d, both significantly shorter than that of the control group (75 d +/- 9 d, both P < 0.01). CONCLUSION: The down-regulation of CacyBP can promote the proliferation of gastric cancer cells and aggravate their malignant behavior.