Ding Lei, Chen Xiaoping, Jing Kai, Wang Haiping, Zhang Wanguang
Department of Hepatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
J Huazhong Univ Sci Technolog Med Sci. 2006;26(1):75-8. doi: 10.1007/BF02828043.
In order to investigate the inhibitory effects on the vascular endothelial growth factor (VEGF) expression and cell growth in hapatocellular carcinoma (HCC) by blocking HIF-1alpha and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides (ASODN) were designed to block HIF-1alpha and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group (P > 0.05), but there was significant difference between ASODN group and control group (P < 0.05). At a concentration of 10 micromol/L ASODN, the difference was very significant (P < 0.01). Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 micromol/L. the integral gray levels and RNA odds were 59743.2 +/- 10412.5 and 0.783 +/- 0.032 in ODN group, and 38694.5 +/- 10925.1 and 0.468 +/- 0.015 in ASODN group, respectively, with the difference being very significant (P < 0.01). Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1alpha action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.
为了通过阻断血管内皮生长因子（VEGF）启动子中的缺氧诱导因子-1α（HIF-1α）和Smad3结合位点，研究其对肝细胞癌（HCC）中VEGF表达及细胞生长的抑制作用，设计了反义寡脱氧核苷酸（ASODN）来阻断VEGF启动子中的HIF-1α和Smad3结合位点。分别将不同浓度的ASODN和寡脱氧核苷酸（ODN）转染至肝癌细胞中。采用SABC法、蛋白质免疫印迹法及逆转录-聚合酶链反应（RT-PCR）技术检测VEGF mRNA和蛋白的表达，并运用MTT法测定其对上清液刺激的肝癌细胞VEGF表达及细胞生长的抑制作用。免疫组织化学结果显示，将不同浓度的ODN和ASODN分别与肝癌细胞共接种48小时后，ODN组与对照组VEGF蛋白表达差异无统计学意义（P>0.05），而ASODN组与对照组差异有统计学意义（P<0.05）。当ASODN浓度为10μmol/L时，差异非常显著（P<0.01）。蛋白质免疫印迹法及RT-PCR结果显示，10μmol/L浓度处理48小时后，ODN组的积分吸光度值及RNA相对含量分别为59743.2±10412.5和0.783±0.032，ASODN组分别为38694.5±10925.1和0.468±0.015，差异均非常显著（P<0.01）。反义ODN可呈浓度依赖性抑制肝癌细胞生长。研究得出结论，针对VEGF启动子中HIF-1α作用位点的反基因技术可抑制VEGF表达并抑制肝癌细胞生长，反基因技术有望成为肝癌抗血管生成的新型基因治疗工具。
