Research On The Roles And Mechanism Of CHPF In The Development Of Osteosarcoma

Objectives:The objective of our research was to explore and verify the function of chondroitin polymerization factor(CHPF)on the proliferation,invasion and apoptosis of human osteosarcoma through in vivo and in vitro experiments,and to explore and figure out the possible downstream regulatory mechanisms.Methods:1.Osteosarcoma cell lines U-2OS and MNNG/HOS were transfected by sh CHPF and sh Ctrl lentiviruses,and their knockdown efficiency was tested with WB and q-PCR.2.Clone formation assay and MTT assay were used to observe the proliferation of osteosarcoma cell lines after CHPF knockdown.3.Flow cytometry and human apoptosis chip array were used to observe the apoptosis and related protein expression of osteosarcoma cell lines after CHPF knockdown.4.Trans-well and Wound-Healing experiments were performed to observe the invasion of osteosarcoma cell lines after CHPF knockdown.5.The effect of CHPF on osteosarcoma cells was evaluated by establishing a subcutaneous tumorigenesis model.6.To explore the mechanism of CHPF regulating the development of osteosarcoma by WB and flow cytometry.Results:1.The results of WB and q PCR showed that the sh CHPF and sh Ctrl osteosarcoma cell lines U-2OS and MNNG/HOS were constructed successfully.2.The number of clones and proliferation of osteosarcoma cell lines decreased significantly after CHPF knockdown.3.After CHPF knockdown,the apoptosis ratios of osteosarcoma cell lines(U-2OS and MNNG/HOS)had significant increase.Furthermore,the expression levels of apoptosis-related proteins like Bax,Bad,Caspase3,Caspase8,CD40 L,Fas,IGFBP-4,P27,p53 and SMAC were significantly up-regulated.4.The migration and invasion ability of osteosarcoma cell lines decreased significantly after CHPF knockdown.5.The results of tumorigenic experiment in nude mice showed that the tumorigenic weight and volume of osteosarcoma cells in nude mice decreased significantly after CHPF knockdown.6.The results of WB and flow cytometry suggest that CHPF may promote the apoptosis of osteosarcoma through PI3K/AKT/m TOR signaling pathway.Conclusions:1.Knocking down CHPF can inhibit the proliferation and invasion of osteosarcoma cell and promote its apoptosis.2.Knocking down CHPF could significantly inhibit the tumorigenic ability of osteosarcoma cell in nude mice.3.Knocking down CHPF may promote the apoptosis of osteosarcoma by inhibiting the PI3K/AKT/m TOR signaling pathway.