RNA干扰对肝素酶在肝癌细胞中表达及其肿瘤生长抑制的作用

Silencing of heparanase by siRNA inhibits tumor growth of human hepatocellular carcinoma in vitro and in vivo

目的:采用RNA干扰(RNAi)技术抑制肝癌细胞系HepG2肝素酶(Hpa)的表达,体内观察其对肿瘤生长的抑制作用.方法:以脂质体介导的方法将构建的2个抗Hpa短发卡式RNA(Hpa-shRNA)真核表达载体稳定转染人肝癌细胞株HepG2(高表达Hpa),并设空载体对照组;流式细胞技术鉴定转染稳定性,Western blot及RT-PCR检测肝素酶蛋白和mRNA表达;将干扰组和对照组的肿瘤细胞皮下接种裸鼠,比较各组肿瘤的体质量和体积大小,免疫组化法检测肝素酶在肿瘤组织的表达.结果:与空载体组相比,干扰后的HepG2细胞中Hpa mRNA和蛋白表达明显降低,干扰组裸鼠的肿瘤生长速度明显变慢,统计结果表明干扰组在抑制肿瘤生长方面与空载体组、空白对照组存在显著差异(P<0.05),两干扰组无显著差异(P>0.05).结论:应用RNAi技术沉默Hpa基因可以有效下调肝癌肿瘤细胞中Hpa mRNA及蛋白的表达,抑制体内肿瘤生长.

AIM： To investigate the inhibitory effect of siRNA on heparanase expression in HepG2 cells in vitro and in vivo. METHODS： Human hepatoceUular carcinoma （HCC） cell line HepG2, which has naturally high levels of endogenous heparanase, was transfected with a plasmid expressing siRNA directed against human heparanase mRNA （experimental groups）. An empty vector was designed as a control. The stability of transfection was analyzed by flow cytometry. RT-PCR and Western blotting were used to confirm the efficient silencing of heparanase gene expression. Cells transfected with siRNA vectors were inoculated subcutaneously into nude mice to test the proliferation of tumor cells. Heparanase expression in tumor was tested by immunohistochemistry. RESULTS： Compared with cells transfected with empty vector, HepG2 cells transfected with the anti-heparanase siRNA vectors had a significant decrease in heparanase expression. In vivo, tumors produced by cells transfected with the anti-heparanase siRNA vectors grew much slower than those arising from cells transfected with the empty vector. There were significant differences in the inhibitory effect on tumor growth between the experimental and control groups （P 〈 0.05）, but there were no significant differences between the experimental groups. CONCLUSION： Down-regulating the expression of heparanase by RNA interference can significantly reduce the expression of heparanase in HepG2 human HCC cells, which leads to inhibition of proliferation of HCC cells in vivo.