The Influence Of STGC3 Gene Overexpression On The Radiosensitivity Of CNE2 Cells

OBJECTIVE: Restoring the overexpression of STGC3 to investigate the radiosensitivity of CNE2 cells and potential mechanisms,providing a new experimental basis for elevating the efficiency of NPC radiation therapy.METHODS: 1.CNE2-RTP-STGC3-his was established by lentivirus transfection.2.The experiments were divided into two groups:(1)The irradiation group: CNE2-RTP-STGC3-his cell groups,CNE2-RTP-his cell groups and CNE2 groups were irradiated with 6MV X-ray for 2 minutes,the total dose is 4Gy.(2)The non-irradiated group: CNE2-RTP-STGC3-his cell groups,CNE2-RTP-his cell groups and CNE2 groups.3.The survival rate of irradiated cells was detected by CCK-8 method.4.Transwell experiment was used to reveal the migration and invasion ability of cells that irradiated by X-ray.5.SDS-PAGE and Protein Mass Spectrometry were used to analyze the changes in protein expression after irradiation.6.Detecting the expression level of partial difference protein and autophagy-related protein LC3,p62,p-S6 and p-ULK1 by Western Blot.RESULTS: 1.The CNE2-RTP-STGC3-his cell line and the CNE2-RTP-his cell line were successfully established by our previous research group.The detection of red fluorescence protein(RFP)showed that RFP was expressed in transgene groups and empty vector groups under fluorescence microscope.Western Blot showed that the STGC3 protein in CNE2-RTP-STGC3-his was highly expressed.(p<0.05)2.CCK-8 method confirmed the survival rate of the CNE2-RTP-h STGC3-his cell line had a significant decrease compared to the CNE-RTP-his cell line after X-ray irradiation(p<0.05).3.Transwell assay revealed that both the number of cell migration and invasion of the CNE2-RTP-STGC3-his cell line were less than that of the control group after X-ray irradiation(p<0.05).4.The results of SDS-PAGE electrophoresis,protein identification and Mascot database analysis showed that the protein expression of 14-3-3,TUBA1 B and calnexin in the irradiation group all had an increase compared to the non-irradiated group.5.Western Blot assay showed that the expression of 14-3-3 and autophagy-related protein LC3-II were decreased in the CNE2-RTP-STGC3-his cell line(p<0.05).By contrast,p62,p-S6 and p-ULK1 were increased(p<0.05).CONCLUSIONS: 1.Overexpression of STGC3 gene could enhance the radiosensitivity of CNE2 cells,which possibly via the inhibition of autophagy that induced by irradiation.