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。
Objective：To construct Krtippel-like factor 8 （KLF8）expression plasmid pc DNA3.1-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,peDNA3.1 ,pGPU6/GFP/Neo-KLF8 and pGPU6/GFP/Neo-ShNC plasmid were transfeeted into human SMMC7721 cells separately, mRNA levels of hypoxia-inducible factor（HIF）1-α,vascular endothelial growth factor （VEGF）, angiopoietin 1 （Angl）, 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 significandy 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 Angl 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 Angl displayed no significant changes （P 〉0.05） in experiment group（transfected with pGPU6/GFP/Neo-KLF8）. Conclusion：KLF8 may regulate angiogenesis in SMMC7721by regulating several angiogenesis related factors such as VEGF, Ang2,HIF1-α and MMP2.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2013年第5期483-488,共6页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81001096/H1617)
重庆市自然科学基金资助项目(编号:CSTC
2010BB5395)
