| Objective: To investigate the role of KLF8in regulating angioge-nesis related factors in SMMC7721.Methods: KLF8interference plas-mid pGPU6/GFP/Neo-KLF8targeting four interference sites was cons-tructed by Genepharma Co, Shanghai, and then screen the most effe-ctive one. RT-qPCR and Western blot were used to confirm the effe-cts on regulating KLF8levels. pcDNA3.1-KLF8, pcDNA3.1, pGPU6/GFP/Neo-KLF8and pGPU6/GFP/Neo-ShNC plasmid were transfectedinto human SMMC7721cells separately.mRNA levels of hypoxia-ind-ucible factor1-α(HIF1-α),vascular endothelial growth factor (VEGF),angiopoietin1(Ang1),angiopoietin2(Ang2),angiopoietin receptor2(Tie2),matrix metallo-proteinase2(MMP2) were investigated by qPCR.Westernblot were used to confirm the expression of VEGF.Results:pcDNA3.1-KLF8significantly up-regulated KLF8expressions(relative expressions:68.8±6.5).The most efficient KLF8interference plasmids pGPU6/GFP/Neo-KLF8was obtained and pGPU6/GFP/Neo-KLF8down-regulatedKLF8expressions(relative expressions:0.31±0.01).Compared with those in control group (transfected with pcDNA3.1), mRNA levels ofVEGF, Ang2, HIF1-α and MMP2were increased(P<0.05) while Tie2and Ang1had no significant changes(P>0.05) in experiment group(transfected with pcDNA3.1-KLF8).Meanwhile, compared with thosein control group (transfected with pGPU6/GFP/Neo-ShNC),mRNA lev-els of Ang2and HIF1-α were decreased (P<0.05) while VEGF, Tie2,MMP2and Ang1displayed no significant changes (P>0.05) in exper-iment group (transfected with pGPU6/GFP/Neo-KLF8).The VEGFprotein expression is consistent with the expression of mRNA.Conclusion: KLF8may regulate angiogenesis in SMMC7721by regu-lating several angiogenesis related factors such as VEGF,Ang2,HIF1-αand MMP2. |
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