Construction of shASPP2 H22 Stable Hepatoma Cell Line and the Effect of ASPP2 Knockdown on Angiogenesis in H22 Xenograft Mice
Objective:To construct mouse shASPP2 H22 stable hepatocarcinoma cell line and observe the effect of ASPP2 knockdown on angiogenesis.Methods:Three shRNA sequences(Y18421,Y18422,Y18423)and one control sequence(GL427NC2)were designed for mouse ASPP2 gene.Recombinant plasmid was constructed by double enzyme digestion(Age Ⅰ and EcoR Ⅰ)and plasmid ligation,colony PCR and sequencing alignment were used to identified it.293T cells were used to package the lentivirus with each recombinant plasmid and determined the titers.H22 cells were transfected with shASPP2 and control lentivirus plasmids,the transfection efficiency was determined by flow cytometry.qRT-PCR,Western Blot were used to observe the interference effect of shASPP2 lentivirus on ASPP2 expression in H22 cells.CCK8 assay was used to observe the effect of ASPP2 knockdown on the proliferation of H22 cells.Western Blot was used to observe the effect of ASPP2 knockdown on the expression and secretion of VEGF in H22 cells and supernatant.The mouse subcutaneous xenograft model of ASPP2 knockdown H22 cells was established by cell injection.The tumor volume was observed by vernier caliper method,the tumor angiogenesis was observed by in vivo laser confocal microscopy,and the expression of VEGF in tumor tissue was detected by Western Blot.Results:The results of double enzyme digestion and colony PCR and sequencing alignment showed that the recombinant plasmids were successfully constructed.The titers of Y18421,Y18422,Y18423 and GL427NC2 were 3.40×108 TU/mL,4.08×108 TU/mL 5.49×108 TU/mL and 1.7×109 TU/mL,respectively.Flow cytometry showed that the transfection efficiency of lentiviral plasmid Y18421,Y18422,Y18423 and GL427NC2 were 86.2％,69.6％,60.8％and 76.9％,respectively.The expression of ASPP2 mRNA and protein in Y18421 cells was significantly lower than that in GL427NC2 cells(P＜0.01,P＜0.05);CCK8 results showed that the proliferation rate of Y18421 cells was significantly increased after cultured 24,48 and 72 h(P＜0.0001,P＜0.001,P＜0.01);The expression of VEGF in Y18421 cell and its supernatant were significantly increased(P＜0.001,P＜0.01,P＜0.05);Compared with GL427NC2 cells,Y18421 cells transplanted tumor volume was significantly increased(P＜0.05),Total vessel length increased significantly(P＜0.05),and the expression of VEGF protein was significantly up-regulated in Y18421 cells(P＜0.05).Conclusion:The mouse shASPP2 H22 stable hepatoma cell line was successfully constructed.ASPP2 knockdown can promote the angiogenesis of mouse H22 cell transplantation tumor by promoting the expression of VEGF.
H22 cellsTumor protein p53-binding protein 2Vascular endothelial growth factorAngiogenesis
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