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

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.