The effects of ANGPTL4 gene on the proliferation and migration of osteosarcoma 143B cell in vitro and in vivo

Abstract

Objective To construct a recombinant adenovirus with angiopoietin-like 4 and siangiopoietin-like 4 genes by modified AdEasy system,and investigate the effects of ANGPTL4 gene on the proliferation and migration of osteosarcoma 143B cells in vitro and in vivo.Methods The recombinant adenovirus with ANGPTL4 and siANGPTL4 genes was constructed by modified AdEasy system,and the viral titre was determined by limiting dilution assay.After 143B cells were transfected by recombinant adenovirus,the transfected efficiency of Ad-ANGPTL4 and Ad siANGPTL4 was identified by RT-PCR method through detecting the relative expression levels of the genes.The effects of ANGPTL4 gene on proliferation and migration of the osteosarcoma cell line 143B were determined by Crystal Violet staining,cell count and wound healing assay in vitro.Moreover,the transfected 143B cells were injected into the right tibial medullary cavity of the nude mouse to establish osteosarcoma animal model.The effects of ANGPTL4 gene on osteosarcoma growth were monitored by Xenogen bioluminescence imaging system in vivo.Results The recombinant adenovirus with ANGPTL4 and siANGPTL4 genes was constructed successfully and the viral titre was 1.2 × 1011-2.6 × 1011 pfu/ml.After transfected,the relative expression intensities of ANGPTL4 gene in 143B cells were 157.39 ± 9.87,156.90± 8.95,185.29 ± 8.24,and 129.28 ± 7.28 in negative control,RFP,ANGPTL4 and siANGPTL4 groups,respectively,and the differences were significant (P＜ 0.05).The proliferation and migration abilities of 143B cells were improved by over expression of ANGPTL4 gene and reduced by ANGPTL4 gene silencing (P＜ 0.05).The nude mice model with osteosarcoma was estabilished successfully.The osteosarcoma with ANGPTL4 over expression grew rapidly,however,the growth of the tumor was inhibited obviously by low expression of ANGPTL4(P ＜0.05).Conclusions Recombinant adenovirus carrying ANGPTL4 and siANGPTL4 genes could be constructed by AdEasy system with high titre and efficient transfection.It can promote or inhibit the proliferation and migration of 143B cells in vitro and in vivo by up-regulating or silencing ANGPTL4 gene expression. Key words: Osteosarcoma; Adenovirus; Angiopoietin-like 4