Effect Of STGC3 On Radiosensitivity Of CNE2 Through AMPK/mTOR

OBJECTIVE: In our previous study,we found that restoring the expression of STGC3 gene in CNE2 cells can decrease the protective autophagy of CNE2 cells and enhance the sensitivity of radiotherapy,while the specific mechanism is still unclear.Based on the preliminary results,we intend to explore the effect of STGC3 gene mediated by AMPK/mTOR signaling pathway on the radiosensitivity of CNE2 cells.METHODS:(1)Construction of plasmid(GV147,completed by the previous research group).(2)Empty vector plasmid(RFP)and STGC3 high expression plasmid(RFP-STGC3)were transfected into CNE2 cells using liposome transient transfection technology,divided into CNE2 group,CNE2-RFP group,CNE2-RFP-STGC3 group,observe fluorescent protein expression of each group by fluorescence microscope,Western Blot to detect STGC3 protein expression.(3)After radiation treatment,we use Western Blot to detect AMPK,p-AMPK,mTOR,p-mTOR,LC3 Protein expression.(4)AMPK is an upstream signaling molecule of mTOR,which plays a negative role in regulating mTOR.The activation of mTOR can inhibit the occurrence of autophagy.AMPK activator metformin(MET) and mTOR inhibitor rapamycin(RAPA))Treat the CNE2-RFP-STGC3 group to interfere with the occurrence of autophagy separately,divided into CNE2 group,CNE2-STGC3 group,CNE2-RFP-STGC3 group,CNE2-RFP-STGC3 + MET group,CNE2-RFP-STGC3 + RAPA group.At the same time,radiation treatment was performed.Western Blot was used to detect the expression of AMPK,p-AMPK,mTOR,p-mTOR,and LC3 proteins,and the interference effect was analyzed.(5)CCK-8 test was used to detect cell viability of each group of cells,and scratch test was used to detect cells of each group Migration ability,plate cloning test The colony-forming ability of the cells of each group was measured,and the sensitivity of the cells of each group to radiotherapy was evaluated.RESULTS:(1)After plasmid sequencing,the plasmid was successfully constructed.(2)The empty vector plasmid(RFP)and STGC3 high expression plasmid(RFP-STGC3)were transfected into CNE2 cells.Under the fluorescence microscope,the expression of red fluorescent protein was found in CNE2-RFP and CNE2-RFP-STGC3 groups.The expression of fluorescent protein and high expression efficiency proved that RFP and RFP-STGC3 were successfully transferred into CNE2 cells.Western Blot test results showed that STGC3 protein expression in CNE2-RFP-STGC3 group was significantly higher than that in CNE2-RFP group and CNE2 group(p <0.05),indicating that the STGC3 high-expressing CNE2 cell line(CNE2-RFP-STGC3)and the empty vector CNE2 cell line(CNE2-RFP)were successfully constructed.(3)After the CNE2-RFP-STGC3 group,CNE2-RFP group,and CNE2 group were treated with radiation,Western Blot test results showed that the protein expressions of p-AMPK in the CNE2-RFP-STGC3 group were significantly lower than those in the CNE2-RFP and CNE2groups(p <0.05),indicating that STGC3 could inhibit the activation of AMPK.The expression of p-mTOR protein was significantly higher than that of CNE2-RFP group and CNE2 group(p <0.05),indicating that STGC3 can promote mTOR activation.The expression of LC3-II protein in CNE2-RFP-STGC3 group was lower than that in CNE2-RFP group and CNE2 group(p <0.05),indicating that STGC3 can interfere with the occurrence of autophagy.(4)The CNE2-RFP-STGC3 group was treated with MET and RAPA separately,and radiation treatment was performed simultaneously.After 24 hours in culture,Western Blot test results showed that the protein expression level of p-AMPK in the CNE2-RFP-STGC3 + MET group was up-regulated compared to CNE2-RFP-STGC3(p <0.05),indicating that MET interfered with the negative regulation of STGC3 on AMPK and increased AMPK phosphorylation.The expression level of p-mTOR protein in CNE2-RFP-STGC3 + RAPA group was lower than that of CNE2-RFP-STGC3(p <0.05).It is shown that RAPA antagonizes STGC3 and inhibits mTOR phosphorylation.The protein expression levels of LC3-II in CNE2-RFP-STGC3 + MET group and CNE2-RFP-STGC3 + RAPA group are higher than those in other CNE2-RFP-STGC3 groups(p <0.05).It shows that the cell autophagy level of CNE2 cells with high expression of STGC3 increased after MET and RAPA intervention.(5)CCK-8 suggested that the cell viability of CNE2-RFP-STGC3+ MET group and CNE2-RFP-STGC3 + RAPA group was increased after radiotherapy treatment.CNE2-RFP-STGC3 group increased(p<0.05).Scratch test suggested that CNE2-RFP-STGC3 + MET group and CNE2-RFP-STGC3 + RAPA group had higher cell migration ability than CNE2-RFP-STGC3 group(p <0.05)The plate cloning test showed that the cell cloning ability of the CNE2-RFP-STGC3 + MET group and the CNE2-RFP-STGC3 + RAPA group was increased compared with the CNE2-RFP-STGC3 group(p < 0.05),suggesting that MET and RAPA can antagonize the effects of STGC3 on the migration ability,colony formation of CNE2 cells,and reduce the radiosensitivity of CNE2 cells.CONCLUSIONS:The expression of STGC3 in CNE2 cells can inhibit the autophagy mediated by AMPK/mTOR signaling pathway and enhance the sensitivity of CNE2 cells to radiotherapy.