2334 - LncRNA SNHG17/IGF2BP1/AURKA Axis Promotes Radiation Resistance in Non Small-Cell Lung Cancer by Regulating Autophagy
Presenter(s)
F. Chen1, W. Liu2, J. Wang3, and Y. Mo1; 1Department of Radiation Oncology, The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China, 2Department of Radiation Oncology, The Second Affiliated Hospital of Guangxi Medical University, Nanning, China, 3The Second Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China
Purpose/Objective(s): To investigate the mechanism of LncRNA SNHG17 regulating autophagy in NSCLC radioresistance through IGF2BP1/AURKA axis.
Materials/Methods: 1. The expression of SNHG17 in the above cell lines was detected by RT-qPCR. 2. The effect of SNHG17 on cell proliferation was detected by EdU and plate colony formation assay. 3. Clone formation assay and?-H2AX focus assay were used to explore the effect on cell radiosensitivity after targeted silencing of SNHG17. 4. Changes in the level of cellular autophagic activity and its relationship to changes in radiosensitivity caused by SNHG17-depletion was explored. 5. Explore the effect of SNHG17 on AURKA mRNA stability by mRNA stability experiments. 6. Explore the binding relationship of IGF2BP1 with SNHG17 and AURKA by RIP-qPCR, and MeRIP-qPCR experiment was used to analyze the m6A modification of AURKA. 7. Rescue experiments were applied to verify whether SNHG17 exerts biological effects dependent on AURKA. 8. Mice experiments were performed to investigate the effect of combination treatment on tumor growth.
Results: RT-qPCR showed that the expression of SNHG17 in NSCLC cell lines was significantly higher than that in normal lung epithelial cells. 2. After silencing SNHG17, the growth and proliferation of NSCLC cell lines were inhibited. 3. Western blot showed that the protein expression of AURKA was decreased after knocking down SNHG17, clonogenic survival assays showed that inhibition of SNHG17 obviously increased the radiosensitivity of A549 and H460 cells. Additionally, ?-H2AX focis were significantly increased in the combination of X-ray and SNHG17 inhibition groups compared with that in X-ray treatment alone groups. 4. After knocking down SNHG17, cytotoxic autophagy was enhanced, and silencing SNHG17 combined with radiotherapy, autophagy enhancement was more obvious; silencing SNHG17 combined with the autophagy inhibitor, Chloroquine (CQ), was able to partially reverse the radiosensitization effect induced by the knockdown of SNHG17. 5. mRNA stability experiments confirmed that down-regulation of SNHG17 enhanced the decay of AURKA mRNA in NSCLC cells. 6. We found that SNHG17 interacts with IGF2BP1 and has potential binding sites using catRAPID database. RIP qPCR experiments confirmed the direct interaction between IGF2BP1, SNHG17, and AURKA. MeRIP qPCR experiments showed that IGF2BP1 mainly enhances the stability of AURKA mRNA through m6A modification. 7. Overexpression of AURKA after knockdown of SNHG17 partially reversed the enhancement of autophagic activity and radiosensitizing effect induced by SNHG17 silencing. 8. In vivo experiments confirmed that silencing SNHG17 combined with radiotherapy significantly inhibited the growth of transplanted tumors.
Conclusion: Through the above research, we found that SNHG17 binds to IGF2BP1, promoting IGF2BP1 recognition of m6A on AURKA mRNA, increasing mRNA stability and AURKA expression, inhibiting autophagic cell death in NSCLC cells, and leading to radiation resistance.