RNAi沉默Cripto基因对结肠癌细胞血管内皮生长因子的抑制

Effects of inhibition of Cripto gene siRNA on vascular endothelial growth factor of colon cancer cell line LS-174T

目的:研究Cripto基因对结肠癌细胞血管内皮生长因子的影响。方法:设计合成针对Cripto基因的小干扰RNA(small interfering RNA,siRNA),转染结肠癌LS-174T细胞。细胞分4组:空载对照组(对照组)和不同浓度(3.125、6.25和12.5 nmol/L)的siRNA组。分别于转染24、48、72 h后收集细胞,以实时定量PCR检测结肠癌细胞CriptomRNA,分别采用Northern blotting和免疫荧光标记法检测癌细胞VEGF mRNA和蛋白表达。分别收集转染72 h的12.5 nmol/L组和对照组细胞,接种于裸鼠背部。30 d后处死裸鼠,收集裸鼠肿瘤组织,对组织VEGF进行免疫组化染色,计算染色平均强度。结果:实时定量PCR检测显示,转染组CriptomRNA被有效地抑制,且呈浓度和时间依赖性。转染组细胞VEGF蛋白和mRNA明显低于对照组。裸鼠肿瘤免疫组化分析显示,转染组VEGF平均染色强度明显低于对照组。结论:Cripto基因参与了结肠癌组织血管生成的调控。采用siRNA下调Cripto基因表达,可抑制结肠癌组织血管生成。

AIM： To study the effects of Cripto gene on vascular endothelial growth factor （VEGF） of colon carcinoma cells. METHODS： Cripto siRNA was designed and constructed. Colon cancer LS - 174T cells were divided into 4 groups： control group and different dose （3. 125, 6. 25 and 12. 5 nmol/L） of siRNA groups. After transfected for 24, 48 and 72 h, colon cancer ceils were harvested to carry on the next tests. Expression of Cripto mRNA was determined with real -time PCR, and immunofluorescence isothiocyanate （FITC） labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF, respectively. The cells in control group and cells transfected with 12. 5 nmol/L siRNA were inoculated into nude mice respectively. 30 days after inoculated, the mice of two groups were executed, and immunohistochemical （ICH） assay was used to evaluate the VEGF protein of mice tumor. RESULTS： siRNA down - regulated the Cripto mRNA in a dose and time dependent manner. Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner. Compared to control, the expression of VEGF protein from ICH assay was lowered significantly （ P 〈 0.05）. CONCLUSION ： Cripto gene might contribute to the regulation of angiogenesis of colon carcinoma. The down - regulation of Cripto gene by siRNA can suppress angiogenesis of human colon cancer.