干扰 Krüppel 样因子 8表达对肝癌 SMMC7721细胞中血管生成相关因子的调控作用

Regulation of altered Krüppel-like factor 8 expressions on angiogensis ralated factors of hepatocelluar carcinoma SMMC7721

摘要:

目的：构建Krüppel样因子8（Krüppel-like factor 8，KLF8）的真核表达质粒及干扰质粒，考查KLF8对人肝癌SMMC7721 细胞中血管生成相关因子的调控作用。方法：构建KLF8的真核表达质粒pcDNA3.1-KLF8，测序确认。构建靶向不同KLF8干扰位点的干扰质粒pGPU6/GFP/Neo-KLF8，筛选出干扰效率最高的pGPU6/GFP/Neo-KLF8。将pcDNA3.1-KLF8、相应的对照质粒 pcDNA3.1，pGPU6/GFP/Neo-KLF8、相应的对照质粒pGPU6/GFP/Neo-ShNC 分别转染人肝癌SMMC7721细胞，共４组。实时荧光定量PCR及Western blot检测KLF8的表达，qPCR检测各组细胞中低氧诱导因子（hypoxia-inducible factor，HIF）1-α、血管内皮生长因子（vascular endothelial growth factor，VEGF）、血管生成素（angiopoietin，Ang）1、Ang2、血管生成素受体Tie2及基质金属蛋白酶（matrix metallo-proteinase，MMP）2的mRNA表达。结果：pcDNA3.1-KLF8显著上调KLF8表达（相对表达量为68.8±6.5），pGPU6/GFP/Neo-KLF8下调KLF8的表达（相对表达量 0.31±0.01）。转染pcDNA3.1-KLF8组与其对照组相比，VEGF、Ang2、HIF1-α及MMP2 mRNA表达增加（P<0.05），血管生成素受体Tie2、Ang1的mRNA表达无差异（P ＞0.05）；转染pGPU6/GFP/Neo-KLF8组与其对照组相比，Ang2和HIF1-α表达降低（P<0.05），VEGF、血管生成素受体Tie2、Ang1及MMP2的表达无差异（P ＞0.05）。结论：在肝癌中，KLF8可能通过调控VEGF、Ang2、HIF1-α及MMP2来调控血管生成。

Abstract:

Objective：To construct Krüppel-like factor 8（KLF8）expression plasmid pcDNA3.1-KLF8 and KLF8 interference plasmid pGPU6/GFP/Neo-KLF8 and to investigate role of KLF8 in regulating angiogenesis related factors in SMMC7721. Methods：KLF8 ex－pression plasmid pcDNA3.1-KLF8 and KLF8 interference plasmid pGPU6/GFP/Neo-KLF8 targeting four interference sites were con－structed and then were screened for the most effective one. Real-time quantitative PCR and Western blot were used to confirm the effects on regulating KLF8 levels. pcDNA3.1-KLF8，pcDNA3.1，pGPU6/GFP/Neo-KLF8 and pGPU6/GFP/Neo-ShNC plasmid were transfected into human SMMC7721 cells separately. mRNA levels of hypoxia-inducible factor（HIF）1-α，vascular endothelial growth factor（VEGF），angiopoietin 1（Ang1），angiopoietin 2（Ang2），angiopoietin receptor Tie2，matrix metallo-proteinase（MMP）2 were investigated by qPCR. Results：Sequencing showed that KLF8 eukaryotic expression plasmids pcDNA3.1-KLF8 was successfully constructed and pcDNA3.1-KLF8 significantly up-regulated KLF8 expressions（relative expression：68.8±6.5）. The most efficient KLF8 interference plasmids pGPU6/GFP/Neo-KLF8 was obtained and pGPU6/GFP/Neo-KLF8 down-regulated KLF8 expressions（relative expression：0.31±0.01）. Compared with those in control group，mRNA levels of VEGF，Ang2，HIF1-α and MMP2 were in－creased（P<0.05） while angiopoietin receptor Tie2 and Ang1 had no significant changes（P >0.05）in experiment group（transfected with pcDNA3.1-KLF8）. Meanwhile，compared with those in control group（transfected with pGPU6/GFP/Neo-ShNC），mRNA levels of Ang2 and HIF1-α were decreased（P<0.05）while VEGF，angiopoietin receptor Tie2，MMP2 and Ang1 displayed no significant changes（P >0.05） in experiment group（transfected with pGPU6/GFP/Neo-KLF8）. Conclusion：KLF8 may regulate angiogenesis in SMMC7721 by regulating several angiogenesis related factors such as VEGF，Ang2，HIF1-α and MMP2.