The Role And Mechanism Of Axon Guidance Factor SLIT2 And Its Specific Receptor ROBO1 In Proliferation And Glucose Metabolism Of Osteosarcoma

Objective:To investigate the role of SLIT2/ROBO1 in the proliferation and metabolic reprogramming of osteosarcoma(OS)and the related molecular mechanism was also explored.Methods:Firstly,the relationship between SLIT2/ROBO1 and the prognosis of osteosarcoma patients was analyzed based on the R2 database.Secondly,the relative expression of mRNA and protein of SLIT2 and ROBO1 was detected in the osteosarcoma cell lines and osteoblast cell line.Then,the expression patterns of ROBO1 and SLIT2 in osteosarcoma and osteochondroma tissue specimens by IHC staining.Then,small inference RNA targeting SLIT2 or ROBO1 gene were transfected into Saos-2 and U-2OS cells,the efficiency was verified by real-time PCR and Western blot.The effect of SLIT2/ROBO1 on the growth of osteosarcoma cells in vivo and vitro was detected by CCK-8,clone formation,apoptosis assay and mouse xenograft model.Seahorse was used to investigate whether the SLIT2/ROBO1 axis was linked to the Warburg effect in OS.Finally,a ChIP assay and luciferase reporter assay were used to explore the molecular mechanism that SLIT2/ROBO1 contributes to the Warburg effect in OS.Results:Database analysis showed that there was a negative correlation between ROBO1 expression and overall survival in patients with osteosarcoma.Compared to the osteoblast cell line hFOB1.19,We found that SLIT2 and ROBO1 was up-regulated in osteosarcoma cell lines Saos-2 and U-2OS.We found that downregulation of SLIT2 and ROBO1 can effectively promote the apoptosis and inhibit the proliferation ability of U-2OS and MNNG-HOS cell lines.Metabolic flux analysis revealed the contribution of the SLIT2/ROBO1 axis to the Warburg effect in OS.We found SLIT2/ROBO1 regulates the SRC/ERK/c-MYC/PFKFB2 pathway to enhance the Warburg effect and facilitate the progression of OS by ChIP assay and luciferase reporter assay.Conclusions:We found that SLIT2 and ROBO1 was up-regulated in osteosarcoma cell lines and osteosarcoma tissues.SLIT2/ROBO1 axis contributed to the Warburg effect in osteosarcoma through activation of SRC/ERK/c-MYC/PFKFB2 pathway,which promoted the proliferation of OS cells and inhibited their apoptosis.