The Reaserch Of FAM92A1-289’ Effect On The Malignant Behavior Of Human Glioma Cells U251 And Its Mechanism

Background:Gliomas are recognized as highly malignant and intractable cancers and are the most common type of primary brain tumors throughout the world.The complex cellular composition,diffuse invasiveness and capacity to escape conventional therapies have been challenging and have hampered progress towards an effective treatment.Molecular targeted therapy for specific genes can perform specifically killing of tumor cells compared with the conventional therapies,and can treat tumors without significantly impairing the normal function of intracranial cells and greatly reduce the recurrence rate.FAM92A1-289 as a family of transcription FAM92A1 variants was discovered in 2007,which is highly expressed in proliferating tumor cells,stem cells and cancer tissues,especially in the embryonic brain tissue,but lowly in the normal brain.So we speculate that this gene may be closely related to the formation of tumors.In addition,there have been many studies confirming that it is closely related to the malignant behavior of tumors such as kidney cancer,cervical cancer,leukemia,etc.Therefore,in this study,we will investigate the potential target of FAM92A1-289 for the first time,and focus on the relationship between the degree of malignancy and malignant behavior of gliomas.Objective:To investigate the correlation between FAM92A1-289（abbreviated as FAM289）and the malignant degree of glioma cell lines its influence on the malignant behavior of glioma cells and its regulation mechanism.This will provide an experimental basis for future molecular targeted therapy of gliomas against this potential target.Methods:（1）In order to investigate the correlation between FAM289gene and the malignancy degree of glioma cell lines,the expression of FAM289was detected by RT-PCR and Western blot in human glioma cell lines（U251,DBTRG and U87MG）.（2）To test the effect of FAM289 on the malignant behavior of gliomas,the stably overexpressed FAM289 in human glioma cell line U251/289⁺⁺was successful constructed by stably transfection CMV-SP6-TALEN-GFP-289 plasmid and puromycin selection.The glioma cell line U251/289⁺⁺was identified by fluorescence microscopy,RT-PCR and Western blot respectively.RTCA（real-time marker-free dynamic cell analysis）,scratch test and transwell assay,tumor formation in mice and other experiments were used to detect the proliferation,migration,and tumor genesis ability of U251 and U251/289⁺⁺cell lines,respectively.（3）To verify the interaction of FAM289 and Galectin-1 protein in eukaryotic cells,a PCS2-3Flag-Galectin-1 plasmid was constructed and identified,and then transfected alone or together with the CMV-SP6-TALEN-GFP-289 plasmid.The co-transfected form was transfected into U251 cells and co-immunoprecipitation experiments were performed to verify whether the interaction of the above two proteins existed.（4）For analyzing FAM289 and Galectin-1 protein’s interaction approachs,transfactsPCS2-3Flag-Galectin-1andCMV-SP6-TALEN-GFP-289 plasmids into U251 cells in the form of common transfection or transfect alone,nuclear and cytoplasmic protein were respectively immune coprecipitate,to detect the interaction place between FAM289 and Galectin-1 protein.Build an RFP-Galectin-1 plasmid and transfect it with CMV-SP6-TALEN-GFP-289 plasmid into U251 cells in the form of common transfection or transfect alone,fluorescence microscope was used to observe the two kinds of fluorescent protein’s position to analysis Galectin-1andFAM289proteins’locationincells.Transfect PCS2-3Flag-Galectin-1 and CMV-SP6-TALEN-GFP-289 plasmids into U251 cells in the form of common transfection or transfect alone,nuclear and cytoplasmic protein extracted respectively,using Western blot to detect the expression of FAM289 and Galectin-1 protein.Results:（1）The expression of FAM289 was increased in a row in U251,DBTRG and U87MG cell lines.（2）Fluorescence microscopy observed that all cells in U251/289⁺⁺cell lines correctly expressed GFP-289 fusion protein and had good growth status.The expression levels of FAM289 in U251/289⁺⁺cell lines were significantly higher than that in U251 cell lines.In the RTCA experiment,the cultivation of 24h optical density value is similar between the two groups（P>0.05）,U251/289⁺⁺cell line cultivate 48,72,96,120h light density values are higher than U251 cell lines（P<0.05 or<0.01）.The migration rate of U251/289⁺⁺cell lines at 24h and 48h was significantly higher than that of U251（P<0.01 or<0.001）.U251/289⁺⁺and U251 cell lines were respectively（242.0±11.41）and（65.40±7.92）,and the two groups were compared with P<0.01.There was no significant difference between U251/289⁺⁺and U251 cell lines in 10 days,and the tumor volume was significantly higher in U251/289⁺⁺cell lines in 20,30 and 40 days.（3）Immunoprecipitation shows that Galectin-1 protein interacts with FAM289 in cells and can be precipitated and detected.（4）The expression levels of DNMT1,DNMT3B gene and protein in U251/289⁺⁺cell lines were significantly higher than that in U251（P<0.01or<0.001）.The total protein expression of ERK and NF-κB was not significantly different in the two groups,but the phosphorylated protein level was significantly higher in U251/289⁺⁺cell lines than in U251（P<0.01）.There was no significant difference in the expression of Galectin-1 protein in the two groups（P>0.05）.（5）FAM289 and Galectin-1 protein did not detect coprecipitation in cytoplasm.In fluorescence co-localization,FAM289 and galectin-1 protein were both located in cytoplasm and cytoplasm at the same time.The expression level of FAM289 in the cell nucleus was significantly higher than that in the cytoplasm（P<0.01）,while the expression of Galectin-1showed no significant change（P>0.05）.Conclusions:（1）The expression of FAM289 in glioma cells was positively correlated with the malignancy of tumor cells.（2）The over-expression of FAM289 can enhance the proliferation,migration in vitro and tumor formation in vivo of human glioma cells U251.（3）FAM289 may regulate ERK/NF-κB signaling pathway by interacting with galectin-1 to regulate the expression of DNMT family proteins to affect the malignant behavior of glioma.（4）The interaction between FAM289 and Galectin-1 is located in the nucleus,and Galectin-1 can promote FAM289 protein into the nucleus.