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microRNA-9 Acts as a Tumor Suppressor and Enhances Radio Sensitivity in Radio-Resistant A549 Cells by Targeting NRP1
E628
International Journal of Radiation Oncology Biology Physics
were significantly enriched in the biosynthesis of amino acids and cell-cell adhesion, can be identified in population RNA-seq and single-cell RNAseq, while the subclone 2 specific genes which were enriched in the RNA splicing and RNA stabilization, only can be identified in the single-cell RNA-seq. Conclusion: Single-cell RNA-seq reveals taxol resistance subpopulations in the resistant cancer cells. And the subclone specific genes may function as a real taxol resistance related genes, which could provide putative targets against drug-resistant for clinical cancer therapy. Author Disclosure: H. Wu: None. J. Yu: None. S. Chen: None. X. Zhang: None. L. Yang: None. H. Zhang: None. Y. Li: None. Q. Hou: None. Y. Hua: None. M. Jiang: None. C. Wang: None. S. Wu: None.
Conclusion: In our study, our group’s previous study has confirmed that WBI can induce cognitive decline detected by behavioral testing. Moreover, the result of Western Blot clearly showed that Whole-brain radiation activated NMDAR/NFAT3/c4/Bax pathway and induced apoptosis, suggesting that NFATc4/3 transcription is involved in radiation-induced neurogenesis impairment. While the inhibitor, 11R-VIVIT peptide rescued the activation of NFAT3/c4-dependent apoptosis after radiation. Based on these results, we believe that radiation-induced cognitive decline is associated with NMDAR-mediated non-conventional apoptotic response. Author Disclosure: M. Xu: None. Q. Fan: None. J. Zhang: None. Y. Tian: None.
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NFAT3/c4-Mediated Excitotoxicity is Involved in Radiation-Induced Cognitive Decline M. Xu,1 Q. Fan,2 J. Zhang,3 and Y. Tian4; 1The Second Affiliated Hospital of Soochow University, Suzhou, China, 2Institute of Radiotherapy & Oncology, Soochow University, 3Suzhou Key Laboratory for Radiation Oncology. San Xiang Road No. 1055, Suzhou 215004, China, Suzhou, China, 3Dept. of Radiotherapy & Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, China, 4Department of Radiation Oncology, Second Affiliated Hospital of Soochow University, Institute of Radiotherapy & Oncology, Soochow University, Suzhou Key Laboratory for Radiation Oncology Suzhou, Suzhou, China
microRNA-9 Acts as a Tumor Suppressor and Enhances Radio Sensitivity in Radio-Resistant A549 Cells by Targeting NRP1 L. Xue and K. Xiong; Peking University Third Hospital, Beijing, China
Purpose/Objective(s): Radiotherapy, an indispensible treatment for head and neck tumor, may cause progressive brain injury, especially injury to the hippocampus, an area important for learning and memory, compromising the quality of life for survivors. However, little is known about the precise molecular pathways for the decline in cognitive function. Recently, the Nmethyl-D-aspartate receptor (NMDAR) is widespread in the CNS and is associated with the occurrence of excitotoxic neuronal cell death. Moreover, nuclear factor of activated T cells isoform 4 (NFAT3/ c4) signaling pathway is vital to excitotoxic hippocampus cell apoptosis induced by radiation. In this regard, it has been assumed that radiation-induced hippocampal-related cognitive dysfunction is associated with NMDAR/NFAT3/c4/Bax pathway. Materials/Methods: SpragueeDawley male rats(21-day old) weighing z50-60g were divided into four groups: the sham group (Sham), the sham with 11R-VIVIT peptide injection (Sham +11R-VIVIT peptide), the irradiation group (IR), the irradiation and 11R-VIVIT peptide injection group (IR +11R-VIVIT peptide), to receive the whole brain irradiation (WBI) treatments with a single dose of 0 (control)or 20Gy by using 4 MeV electron beam. The animals were sacrificed and the hippocampus were removed at 1h, 3h, 6h, 12h, 1d, 3d after irradiation (nZ3 in each group for each time point). Then we use Western blot method to detect the expression of NMDAR subunits (NR2A, not NR1 or NR2B), calcineurin, NFATc4/3, GSK-3b, and apoptosis marker Bax in the rat hippocampal. Results: The results of Western blot indicated that WBI significantly increased NMDAR subunits expression at 6h post-radiation compared with the sham group. While expression levels of CaN and GSK-3b had no difference. With stimulate of radiation, NFAT3/c4 rapidly accumulated in the nucleus, then promoted expression of the apoptosis marker Bax and decreased level of the anti- apoptosis marker Bcl-2. The treatment of 11RVIVIT peptide prevented nuclear accumulation of NFAT3/c4, then inhabited the appearance of the apoptosis marker Bax.
Purpose/Objective(s): Radiotherapy is commonly used to treat lung cancer but does not completely kill cancer cells attributing to the radiotherapy resistance which often happens in non-small cell lung cancer (NSCLC). To date, the molecular mechanism of radio-resistance is still unclear. Neuropilin 1(NRP1), a co-receptor for VEGF, was proved correlated with radio-resistance of NSCLC cells via the VEGF-PI3K-NF-kB pathway in our previous study. We hypothesized that certain miRNAs may play crucial roles in radio-sensitivity by regulating NRP1. Materials/Methods: NSCLC cell line A549 and A549 radio-resistant cell lines were used to perform the in vitro assay. The potential target gene of a certain miRNA was predicted by bioinformatics. The further validation was performed by qPCR and luciferase assay. The expression of the proteins was measured by western blot analysis. Cell viability was evaluated by MTT assay. Cell-cycle analysis and apoptosis were measured by flow cytometry. The colony formation and transwell migration and invasion assay were applied for evaluating the proliferation and mobility of NSCLC cells upon irradiation. Cells were sham-irradiated or exposed to IR at a dose rate of 1.0 Gy/min (220 kV; 18 mA) by an X-ray generator (Model XRAD320, PXI, Branford, Connecticut, USA). Six-week-old male Balb/c athymic nude mice were exposed to a single 20 Gy dose of X rays at a dose rate of 1.55 Gy/min. Results: Bioinformatics predicted that NRP1 was a potential target of miR-9 and it was validated by luciferase reporter assays and qPCR. Functionally, it was found that miR-9 overexpression increased the radiosensitivity of A549 cells. miR-9 transfected A549 cells showed a decreased proliferation rate, increased apoptosis rate and attenuated migration and invasion ability. Moreover, ectopic expression of miR-9 significantly enhanced the radiosensitivity in vivo as well (the nude mouse with tumor model). The results showed that the growth of tumors treated with miR-9 and irradiation were significantly delayed compared with other groups. Mechanistically, it was found that overexpression of miR-9 inhibited PI3K and phosphorylation of NF-kB, P38 MAPK, Erk1/2 and Akt. Conclusion: These findings suggested that miR-9 overexpression combined with radiotherapy could induce a stronger in vivo anti-tumor effect than radiotherapy alone and indicated it might be a molecular prediction target for radio-sensitivity of NSCLC. Author Disclosure: L. Xue: None. K. Xiong: None.
International Journal of Radiation Oncology Biology Physics
were significantly enriched in the biosynthesis of amino acids and cell-cell adhesion, can be identified in population RNA-seq and single-cell RNAseq, while the subclone 2 specific genes which were enriched in the RNA splicing and RNA stabilization, only can be identified in the single-cell RNA-seq. Conclusion: Single-cell RNA-seq reveals taxol resistance subpopulations in the resistant cancer cells. And the subclone specific genes may function as a real taxol resistance related genes, which could provide putative targets against drug-resistant for clinical cancer therapy. Author Disclosure: H. Wu: None. J. Yu: None. S. Chen: None. X. Zhang: None. L. Yang: None. H. Zhang: None. Y. Li: None. Q. Hou: None. Y. Hua: None. M. Jiang: None. C. Wang: None. S. Wu: None.
Conclusion: In our study, our group’s previous study has confirmed that WBI can induce cognitive decline detected by behavioral testing. Moreover, the result of Western Blot clearly showed that Whole-brain radiation activated NMDAR/NFAT3/c4/Bax pathway and induced apoptosis, suggesting that NFATc4/3 transcription is involved in radiation-induced neurogenesis impairment. While the inhibitor, 11R-VIVIT peptide rescued the activation of NFAT3/c4-dependent apoptosis after radiation. Based on these results, we believe that radiation-induced cognitive decline is associated with NMDAR-mediated non-conventional apoptotic response. Author Disclosure: M. Xu: None. Q. Fan: None. J. Zhang: None. Y. Tian: None.
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3500
NFAT3/c4-Mediated Excitotoxicity is Involved in Radiation-Induced Cognitive Decline M. Xu,1 Q. Fan,2 J. Zhang,3 and Y. Tian4; 1The Second Affiliated Hospital of Soochow University, Suzhou, China, 2Institute of Radiotherapy & Oncology, Soochow University, 3Suzhou Key Laboratory for Radiation Oncology. San Xiang Road No. 1055, Suzhou 215004, China, Suzhou, China, 3Dept. of Radiotherapy & Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, China, 4Department of Radiation Oncology, Second Affiliated Hospital of Soochow University, Institute of Radiotherapy & Oncology, Soochow University, Suzhou Key Laboratory for Radiation Oncology Suzhou, Suzhou, China
microRNA-9 Acts as a Tumor Suppressor and Enhances Radio Sensitivity in Radio-Resistant A549 Cells by Targeting NRP1 L. Xue and K. Xiong; Peking University Third Hospital, Beijing, China
Purpose/Objective(s): Radiotherapy, an indispensible treatment for head and neck tumor, may cause progressive brain injury, especially injury to the hippocampus, an area important for learning and memory, compromising the quality of life for survivors. However, little is known about the precise molecular pathways for the decline in cognitive function. Recently, the Nmethyl-D-aspartate receptor (NMDAR) is widespread in the CNS and is associated with the occurrence of excitotoxic neuronal cell death. Moreover, nuclear factor of activated T cells isoform 4 (NFAT3/ c4) signaling pathway is vital to excitotoxic hippocampus cell apoptosis induced by radiation. In this regard, it has been assumed that radiation-induced hippocampal-related cognitive dysfunction is associated with NMDAR/NFAT3/c4/Bax pathway. Materials/Methods: SpragueeDawley male rats(21-day old) weighing z50-60g were divided into four groups: the sham group (Sham), the sham with 11R-VIVIT peptide injection (Sham +11R-VIVIT peptide), the irradiation group (IR), the irradiation and 11R-VIVIT peptide injection group (IR +11R-VIVIT peptide), to receive the whole brain irradiation (WBI) treatments with a single dose of 0 (control)or 20Gy by using 4 MeV electron beam. The animals were sacrificed and the hippocampus were removed at 1h, 3h, 6h, 12h, 1d, 3d after irradiation (nZ3 in each group for each time point). Then we use Western blot method to detect the expression of NMDAR subunits (NR2A, not NR1 or NR2B), calcineurin, NFATc4/3, GSK-3b, and apoptosis marker Bax in the rat hippocampal. Results: The results of Western blot indicated that WBI significantly increased NMDAR subunits expression at 6h post-radiation compared with the sham group. While expression levels of CaN and GSK-3b had no difference. With stimulate of radiation, NFAT3/c4 rapidly accumulated in the nucleus, then promoted expression of the apoptosis marker Bax and decreased level of the anti- apoptosis marker Bcl-2. The treatment of 11RVIVIT peptide prevented nuclear accumulation of NFAT3/c4, then inhabited the appearance of the apoptosis marker Bax.
Purpose/Objective(s): Radiotherapy is commonly used to treat lung cancer but does not completely kill cancer cells attributing to the radiotherapy resistance which often happens in non-small cell lung cancer (NSCLC). To date, the molecular mechanism of radio-resistance is still unclear. Neuropilin 1(NRP1), a co-receptor for VEGF, was proved correlated with radio-resistance of NSCLC cells via the VEGF-PI3K-NF-kB pathway in our previous study. We hypothesized that certain miRNAs may play crucial roles in radio-sensitivity by regulating NRP1. Materials/Methods: NSCLC cell line A549 and A549 radio-resistant cell lines were used to perform the in vitro assay. The potential target gene of a certain miRNA was predicted by bioinformatics. The further validation was performed by qPCR and luciferase assay. The expression of the proteins was measured by western blot analysis. Cell viability was evaluated by MTT assay. Cell-cycle analysis and apoptosis were measured by flow cytometry. The colony formation and transwell migration and invasion assay were applied for evaluating the proliferation and mobility of NSCLC cells upon irradiation. Cells were sham-irradiated or exposed to IR at a dose rate of 1.0 Gy/min (220 kV; 18 mA) by an X-ray generator (Model XRAD320, PXI, Branford, Connecticut, USA). Six-week-old male Balb/c athymic nude mice were exposed to a single 20 Gy dose of X rays at a dose rate of 1.55 Gy/min. Results: Bioinformatics predicted that NRP1 was a potential target of miR-9 and it was validated by luciferase reporter assays and qPCR. Functionally, it was found that miR-9 overexpression increased the radiosensitivity of A549 cells. miR-9 transfected A549 cells showed a decreased proliferation rate, increased apoptosis rate and attenuated migration and invasion ability. Moreover, ectopic expression of miR-9 significantly enhanced the radiosensitivity in vivo as well (the nude mouse with tumor model). The results showed that the growth of tumors treated with miR-9 and irradiation were significantly delayed compared with other groups. Mechanistically, it was found that overexpression of miR-9 inhibited PI3K and phosphorylation of NF-kB, P38 MAPK, Erk1/2 and Akt. Conclusion: These findings suggested that miR-9 overexpression combined with radiotherapy could induce a stronger in vivo anti-tumor effect than radiotherapy alone and indicated it might be a molecular prediction target for radio-sensitivity of NSCLC. Author Disclosure: L. Xue: None. K. Xiong: None.