Molecular Mechanism Of STGC3 Gene Over Expression Enhancing Radiotherapy Sensitivity Of CNE2 Cells

Objective:Previous studies have shown that restoring the expression of STGC3 gene in CNE2 cells leads to enhanced sensitivity of CNE2 cells to radiotherapy and decreased autophagy,but the mechanism remains unclear.On the basis of previous work,this study determined the difference in protein expression of CNE2 transfection group,and confirmed the molecular mechanism of STGC3 gene enhancing the radiosensitivity of CNE2 cells.Methods:1.Over expression of STGC3 plasmids construction.2.STGC3 gene and Empty vector were respectively transferred into CNE2 cell lines by liposomes,and CNE2 cells without any plasmid were set as the control.The experiment was divided into the two groups: 1)Irradiation groups:CNE2-RFP-STGC3 cells(STGC3 overexpression groups),CNE2-RFP cells(Empty vector group),CNE2 cells,They were all respectively irradiated with the dose of 2Gy、4 Gy、6 Gy、8 Gy by 6MV X-ray;2)Unirradiated groups:CNE2-RFP-STGC3 cells,CNE2-RFP cells and CNE2 cells.3.Morphological changes of cells in the Irradiation groups were observed by microscope.4.Transwell assay was used to detect invasion and migration ability of cells before and after irradiation.5.Expression levels of Autophagy related proteins Beclin1、barkor、vps34 and Apoptosis related proteins Bcl-2 were detected by Western Blot.6.Coomassie Brilliant Blue and Protein Mass Spectrometry were used for detecting the differences in protein expression in the Irradiated groups.Results: 1.STGC3 highly expressed plasmids were successfully constructed.2.STGC3 gene and Empty vector were transiently transferred to CNE2 cell lines to construct CNE2-RFP-STGC3 and CNE2-RFP cell lines: 1)Fluorescence detection: the number of Red Fluorescent Protein(RFP)expression in cells of the CNE2-RFP-STGC3 and CNE2-RFP cell groups were both more than 90% under fluorescence microscope.2)Western Blot analysis: the expression of STGC3 protein in CNE2-RFP-STGC3 group was significantly higher than that in CNE2-RFP group and CNE2 group,and the difference was statistically significant(p<0.05),which could be used for subsequent experiments.3.With the increase of irradiation dose(2Gy、4Gy、6Gy、 8Gy),the observation results of inverted microscope showed that all cells in the irradiation group had typical EMT morphological changes and the CNE2-RFP-STGC3 group changes were particularly significant.4.The results of Transwell experiment revealed that both the migration and invasion ability of CNE2-RTP-hSTGC3-his were lower than those of the control groups after radiotherapy after irradiation(p<0.05).5.Western Blot results realved that the expression levels of Autophagy related proteins of Beclin1、barkor 、vps34 and the Apoptosis related proteins of Bcl-2 in the CNE2-RFP-STGC3 group were all decreased after irradiation(p<0.05).6.Coomassie brilliant blue staining and Protein Mass Spectrometry、Mascot database identification were used to analyze the differences of protein expression and the results showed that the protein expression of Heat shock 70 kDa protein 1 A and Phosphoglycerate kinase 1 in the experimental group all had an decrease compared to the control group as the X-ray dose increased.the protein expression of 60 kDa heat shock protein,mitochondrial had an increase compared to the control group as the X-ray dose increased.Conclusions: 1.Up-regulated STGC3 gene can enhance the radiotherapy sensitivity of CNE2 cells,which is related to the inhibition of radiation-induced autophagy and the promotion of apoptosis.2.Over expression of STGC3 gene in CNE2 cells can decrease the expression of type III PI3K-Beclin1,PGK-1,Hsp70 and Bcl-2 proteins,and increase the expression of Hsp60 protein,resulting in decreased autophagy and increased apoptosis.