The Role Of High Expression Of SPATA5L1 In Tumor Growth,Invasion And Metastasis And Its Regulatory Mechanism

Background and objection : The researchers in our laboratory had previously found that the expression of SPATA5L1 in KB cells of human oral epithelial cancer was significantly increased after proper paclitaxel treated /low-dose ionizing radiation.However,the relevant studies and reports on SPATA5L1 gene are currently very limited,and the effect of the over-expressed gene on the tumor invasion and metastasis had not reported yet.Therefore,the aim of this study was to investigate the role of the over-expressed SPATA5L1 gene in the process of tumor cell growth,invasion,migration and metastasis,as well as the effects on the tumor radiosensitivity and the survival rate of transplanted tumor-bearing mice,to explore its regulation mechanism and to provide a new target for tumor therapy in the future.Method:1.The liposome mediated plasmid transfection and G418-resistant screen were used to establish the over-expressed SPATA5L1 human oral epithelial cancer KB cell line and mouse breast cancer 4T1 cell line with recombinant plasmid pc DNA3.1-humanSPATA5L1 and the pc DNA3.1-mouseSPATA5L1 respectively.The gene over-expression was verified with real-time PCR and western blot.The CCK-8assay was used to detect the effect of over-expressed SPATA5L1 gene on the proliferation and radiosensitivity of tumor cells.Transwell and Matrigel vascular endothelial formation assay were used to investigate the effect of over-expressed SPATA5L1 gene on the tumor cell migration,invasion and angiogenesis in vitro.2.The tumor cells were injected into subcutaneous and footpad of BABL/C nude mice to establish a subcutaneous transplantation tumor as well as a footpad implanted tumor model,respectively.The implanted tumor volume,body weight and survival condition of the tumor-bearing mice were observed.The local implanted tumor growth,lymphatic metastasis and blood metastasis were observed by Micro PET/CT imaging,bioluminescent imaging,anatomical and pathological technique as well as immunohistochemistry technique.3.Based on GC-MS metabonomics and KEGG metabolomics database,the difference of metabolites between the mouse breast cancer 4T1 cell,implanted tumor and plasma of the tumor-bearing mice over-expressed mouseSPATA5L1 and the non-transfected 4T1 cell,implanted tumor and plasma of the tumor-bearing mice.Result:1.The two kinds of over-expressed SPATA5L1 gene cell lines had been established successfully,these were named: pc DNA3.1-humanSPATA5L1 KB cell and pc DNA3.1-mouseSPATA5L1 4T1 cell.Compared with non-transfected cells,the proliferation ability and radiosensitivity of KB cells and 4T1 cells with over-expressed SPATA5L1 gene was significantly increased(p<0.05),the ability of migration,invasion and angiogenesis were decreased significantly(p<0.01).2.Compared with KB as well as 4T1 cell subcutaneous tumor bearing mice,the local tumor growth of the KB as well as 4T1 cells with over-expressed SPATA5L1 increased significantly,the volume of the transplanted tumor increased significantly(p<0.01),no liver metastases were found,tumor metastasis was significantly inhibited(p<0.01),the body weight increased significantly(p<0.01),and the body nutritional status was good,no cachexia was found,and the survival rate increased significantly(p<0.01).Similarly,compared with 4T1 cell footpad tumor bearing mice,after overexpression of mouseSPATA5L1 gene,tumor growth and volume increased,lymph node metastasis and liver metastasis were significantly reduced,and body weight and survival rate increased significantly(p<0.01).3.The results of metabonomics analysis showed that the contents of lactate,serine,threonine and cholesterol decreased in over-expressed mouseSPATA5L1 4T1 cells and xenografts(p<0.01).And the content of urea decreased significantly in plasma of the footpad tumor bearing mice implanted over-expressed mouseSPATA5L1 4T1(p<0.01).Systemic summary and analysis with Metabolomics Pathway Analysis 3.0 and KEGG on-line software,we found that the ammonia acyl transfer RNA(t RNA)biosynthesis,sugar metabolism,glycine,serine and threonine metabolism pathways were affected by over-expressed mouseSPATA5L1.The difference of metabolomics was one of the mechanisms for the decline of tumor migration and invasion ability.In the later stage of tumor bear mice,over-expressed mouseSPATA5L1 could improvecachexia by correcting the abnormal metabolism of protein decomposition and synthesis disorders.Conclusion: The over-expressed SPATA5L1 gene can promote tumor growth in situ,powerfully inhibit the invasion,angiogenesis,lymphatic and blood metastasis of tumor cells.It also enhanced the radiosensitivity of tumor,control and reverse the cachexia of the mice,and improve the survival rate of the tumor-bearing mice.The mechanism may be that the over-expressed SPATA5L1 can reduce the level of glycolysis of tumor cells,decrease the contents of lactic acid intracellular and extracellular microenvironment,increase the protein synthesis and hold back protein decomposition.SPATA5L1 gene may be a new target for tumor therapy,it is potential to develop new therapeutic drug.