Transcription Factor NF-YA Promotes Osteosarcoma Cell Malignant Phenotype Via Up-regulating Fatty Acid Synthase(FASH) Expression In Vitro

Objective:To build and screening raised and lowered the NF- YA gene plasmid of the virus. Discuss the NF- YA in osteoblast and osteosarcoma expression differences of cells, and research the NF- YA gene is through FASN gene expression of osteosarcoma cells(HOS and U2-OS) in vitro proliferation, migration and invasion ability.Method:(1)Conventional culture osteoblast(HOB anywhere) and osteosarcoma cells(HOS and U2-OS), divided into three groups, HOB anywhere, HOS and U2-OS groups. Detection the protein and m RNA of FASN were detected by q RT-PCR and western blot.(2)The U2-OS and HOS cells were transfected with the slow virus vector of Plv/CD NF-YA,Plv/SH NF-YA and Plv NF-YA respectively, each cell is divided into three groups, Plv/CD groups、Plv/SH groups and Plv groups.Detection the ability of cells proliferation and migration was measured by MTT, wound healing and transwell invasion assays. Through the q- PCR and Western blot experiments to detect each group of cells of NF- YA and FASN m RNA and protein expression.Results:(1)The m RNA and protein of NF-YA in U2-OS, HOS and HOB cells were detected by q RT-PCR and western blot. The results showed that both of m RNA and protein of NF-YA in HOB cell were significantly lower than those in U2-OS and HOS cells. It suggested that NF-YA was over-expression in OS cells.(2)The U2-OS and HOS cells were transfected with the slow virus vector of Plv/CD NF-YA, Plv/SH NF-YA and Plv NF-YA, respectivily for 24 h. NF-YA m RNA and protein expression levels in U2-OS and HOS cells were detected by q-PCR and western blot analysis. The expression level of NF-Y in cells transfected with the Plv/CD NF-YA was significantly higher than in those treated with Plv NF-YA,and lower in cells infected with Plv/SH NF-YA than in those infected with Plv NF-YA. These results indicate that build containing encoding of NF-YA slow virus vector transfection of osteosarcoma cells(HOS and U2-OS) is successfully.(3)To investigate whether NF-YA promote FASN expression in OS cells,the U2-OS and HOS cells were transfected with the slow virus vector of Plv/CD NF-YA, Plv/SH NF-YA,Plv NF-YA.The protein and m RNA of FASN were detected by q RT-PCR and western blot. The results revealed that the m RNA and protein of FASN in OS cells treated with Plv/CD NF-YA were significantly higher than in those cells infected with Plv NF-YA. However, both of m RNA and protein of NF-YA were significantly decreased in cells infected with Plv/SH NF-YA compared to those of cells treated with Plv NF-YA. It indicated that increased NF-YA expression could promote FASN expression in OS cells.(4)To elucidate the effect of changes NF-YA expression on the malignant phenotype of OS cells,the U2-OS and HOS cells were treated with the virus vector of Plv/CD NF-YA, Plv/SH NF-YA and Plv NF-YA respectively. The ability of cells proliferation and migration was measured by MTT, wound healing and transwell invasion assays. The results showed that the cell proliferation, migratory and invasive ability treated with Plv/CD NF-YA cells was significantly higher than those cells treated with Plv NF-YA, and reated with Plv/SH NF-YA was lower than those treated with Plv NF-YA. It suggested that elevated NF-YA promotes malignant phenotype of OS cells in vitro.Conclusion:NF-YA was elevated expression in OS cells.NF-YA regulate FASN expression in OS cells.Changes of NF-YA expression alters malignant phenotype of OS cells in vitro,which laid a foundation for the next step research.