慢病毒载体介导siRNA沉默Id-1通过ERK1/2信号通路抑制裸鼠人肝癌移植瘤生长

目的 构建慢病毒表达载体介导siRNA沉默Id-1,观察其对人肝癌HepG2细胞裸鼠皮下移植瘤生长的抑制作用及其对ERK1/2信号通路的影响。 方法 构建合成特异性针对Id-1基因的siRNA慢病毒载体,转染肝癌HepG2细胞系,经半定量RT-PCR鉴定筛选沉默效果最佳的细胞系,于倒置荧光显微镜(×400)下观察转染前后细胞的形态学变化。取细胞浓度为5×10⁶ mL^-1的干扰效率最佳的稳定转染细胞系、稳定转染空载体病毒细胞系及正常HepG2细胞系悬液各0.2 mL,分别注射到转染实验组、阴性对照组及空白对照组的裸鼠右腋皮下,每周测量肿瘤体积及裸鼠体质量,绘制肿瘤生长曲线,28 d后处死裸鼠,制作肿瘤组织标本,行常规病理检查,采用半定量RT-PCR及Western-blot方法检测肿瘤组织Id-1,ERK1/2的mRNA和蛋白的表达水平及p-ERK1/2的蛋白表达水平。 结果 倒置荧光显微镜显示,转染前后细胞形态学变化不明显。经半定量RT-PCR筛选出Id-1基因的siRNA慢病毒载体的最佳细胞系为sh31,与稳定转染空载体病毒细胞系相比,目的基因Id-1的表达降低了80%以上,与未干扰的HepG2细胞相比降低了60%; 转染细胞皮下接种后,转染组最终瘤体大小明显小于阴性对照组和空白对照组(P<0.05)。半定量RT-PCR显示,转染组肿瘤组织的Id-1及ERK1/2 mRNA分别为(0.389±0.058)及(0.475±0.079),均明显低于阴性对照组[(0.845±0.113),(0.977±0.082)]和空白对照组[(0.917±0.083),(0.978±0.056)](均为P<0.05)。Western-blot检测显示,转染组的Id-1及p-ERK1/2蛋白均明显低于阴性对照组和空白对照组(P<0.05)。 结论 Id-1基因特异性siRNA的慢病毒表达载体通过靶向抑制Id-1的表达,明显抑制人肝癌细胞HepG2裸鼠皮下移植瘤的生长,推测Id-1可能通过调节ERK1/2 MAPK信号通路参与肝癌的发生发展。

Inhibitory Effect of Lentiviral Vector for siRNA of Id-1 on HumanTransplanted Carcinoma in Nude Mice

ABSTRACT: Objective To construct a lentiviral vector carrying siRNA to silence Id-1, and observe its inhibition of human hepatocellular carcinoma HepG2 of subcutaneous tumor in nude mice and its effect on ERK1/2 signaling pathway. Methods Lentiviral vectors contained siRNA-Id-1 were constructed, and then were transducted into human hepatocellular carcinoma HepG2 cells. The cell lines with best silencing effect were iderntified by RT-PCR. In vivo experiment was carried out by inoculation of HepG2 cells into nude mice, the tumor growth was measured, the tumor growth curve was mapped, and then the tumor specimens were made. The expression of Id-1, ERK1/2, p-ERK1/2 in mRNA and protein level were examined by Semi-quantitative RT-PCR and Western-blot, respectively. Results RT-PCR identified that sh31 liver cancer HepG2 stable cell lines had the best silence effect, the growth of transfected human hepatocellular HepG2 tumor in the nude mice was also significantly inhibited. Tumor cells were large necrosis and vaculation with homogeneous and red dye performance. RT-PCR results displayed that compared to the control group, the mRNA expression levels of Id-1, ERK1/2, p-ERK1/2 in transfected experimental group were decreased while the difference were statistically significant(P<0.05). Western-blot results showed that the protein expression levels of Id-1, ERK1/2 in transfected experimental group were also decreased, and the difference between the experimental groups and the control group were also statistically significant(P<0.05). Conclusion The results of this study demonstrated that the growth of hepatocellular carcinoma cells is effectively inhibited by transfection of Id-1-RNAi-Lentivirus in vivo, and indicated that Id-1 may influence the development of liver cancer by regulating the ERK1/2 MAPK signaling pathway.