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Unmethylated cytosine-phosphorothioate-guanine (CpG)-containing oligodeoxynucleotides (ODNs) are synthetic DNA sequences that mimic bacterial DNA, and are known to serve as ligands for Toll-like receptor 9 (TLR9). The interaction between a CpG ODNs with TLR9 activates the complex downstream cascade that contributes to exerting its function. In the present study, the results of clonogenic assays demonstrated that the activation of TLR9 by CpG ODNs significantly increased the radiosensitivity of A549 lung cancer cells, with a sensitivity enhancement ratio (SER) of 1.28. When the expression of TLR9 was effectively silenced, CpG ODNs used alone were identified to produce SERs as low as 1.01. Flow cytometry demonstrated that the interaction between TLR9 and CpG ODN 7909 alone did not significantly affect the rate of apoptosis, but may significantly enhance the radiation-induced apoptosis of A549 cells. Western blot analysis revealed that TLR9 activation by CpG ODN 7909 increased the levels of mitogen-activated protein kinase 14, cellular tumor antigen p53, B-cell lymphoma 2 associated X protein and genome polyprotein, and decreased Bcl-2 expression levels, whereas these effects were not observed in CpG ODN 7909-treated cells in which TLR9 was knocked down. These results suggest that CpG ODN 7909 may enhance radiosensitivity through TLR9 activation, and partially via the p53 pathway in A549 lung cancer cells. [ABSTRACT FROM AUTHOR]

MicroRNA-302a-5p (miR-302a-5p) has been implicated in several cancers; however, its role in human non-small cell lung carcinoma (NSCLC) remains unknown. In this study, we showed that miR-302a-5p is downregulated in NSCLC tissues and cell lines. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays showed that overexpression of a miR-302a-5p mimic suppressed NSCLC cell proliferation, which was confirmed by the results of a cell cycle assay. Overexpression of miR-302a-5p also reduced the migration and invasion of NSCLC cells. Additionally, miR-302a-5p overexpression significantly inhibited NSCLC growth and metastasis in a mouse xenograft model. With regard to the underlying mechanism, integrin α6 ( ITGA6 ) mRNA was shown to be a novel target of miR-302a-5p, and overexpression of ITGA6 attenuated the inhibitory effects of miR-302a-5p on the proliferation and migration of NSCLC cells. In clinical NSCLC samples, miR-302a-5p expression was negatively correlated with ITGA6 expression, which was high in the samples. Collectively, these results indicate that miR-302a-5p acts as a tumor suppressor in NSCLC by directly targeting ITGA6 mRNA and may be useful as a theranostic biomarker of NSCLC., Competing Interests: None.

Purpose: The aim of our study was to explore the relationships between the M2 isoform of pyruvate kinase (PKM2) and the sensitivity of human non-small cell lung cancer (NSCLC) cells to docetaxel in vitro., Materials and Methods: With the method of plasmid transfection, we silenced the expression of PKM2 successfully in A549 and H460 cells. Western blotting and real-time PCR were applied to detect PKM2 expression at protein and gene levels. Cell viability was examined by CCK8 assay. Cell cycle distribution and apoptosis were examined by flow cytometry. P21 and Bax were detected., Results: Expression of PKM2 mRNA and protein were significantly decreased by shRNA targeting PKM2. Silencing of PKM2 increased docetaxel sensitivity of human NSCLC A549 and H460 cells in a collaborative manner, resulting in strong suppression of cell viability. The results of flow cytometric assays suggested that knockdown of PKM2 or docetaxel treatment, whether used singly or in combination, blocked the cells in the G2/M phase, which is in consistent with the effect of the two on the expression of p21. Cells with PKM2 silencing were more likely to be induced into apoptosis by docetaxel although knockdown of PKM2 alone can't induce apoptosis significantly, which is in consistent with the effect of the two on Bax expression., Conclusion: The results suggest that PKM2 knockdown could serve as a chemosensitizer to docetaxel in non-small lung cancer cells through targeting PKM2, leading to inhibition of cell viability, increase of cell arrest of G2/M phase and apoptosis., Competing Interests: The authors have no financial conflicts of interest.

Objectives: Irradiation resistance appears as local recurrence and distant metastasis in advanced stages of non-small cell lung cancer (NSCLC). High-dose irradiation combined with immunotherapy improved overall survival and local control of NSCLC. This study explored the underlying molecular mechanism by which the effect of high-dose irradiation plus toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotide (CpG ODN) 7909 on NSCLC., Materials and Methods: NSCLC H460 cells were exposed to constant high-dose irradiation (6.37 Gy) in irradiation (IR) group and the irradiation plus CpG group. Gene expression was assessed using quantitative reverse transcriptase-polymerase chain reaction and Western blot. Knockdown of nuclear factor kappa B (NF-κB) p65 expression was conducted using p65 siRNA., Results: Expression of programmed death-ligand 1 (PD-L1) mRNA was significantly decreased in IR combined with CpG ODN 7909 group compared with the control or IR-alone groups ( P <0.05). TLR9 expression was also obviously increased in the combination group compared with the control ( P <0.05). Moreover, expression of NF-κB p65 was apparently reduced in the combination group compared with the control ( P <0.05). However, expression of PD-L1 was significantly decreased after knockdown of p65 in IR group ( P <0.05), but increased in the combination group ( P <0.05) and slightly increased in CpG ODN-alone group ( P <0.05), which was opposite to that without p65 knockdown group., Conclusion: This study demonstrated that radiotherapy combined with CpG ODN 7909 was able to downregulate PD-L1 expression through inhibition via the NF-κB signaling pathway., Competing Interests: The authors report no conflicts of interest in this work.

Background: Cytosine-phosphate-guanine (CpG) oligodeoxyribonucleotides (ODNs), which induce signaling via Toll-like receptor 9, have recently been suggested to enhance sensitivity to traditional therapies, including chemotherapy, in certain cancer cell lines. This study aimed to define the dose-effect relationship for CpG ODN 1826 in increasing radiosensitivity and its impact on immune function in a mouse model of Lewis lung cancer., Methods: The tumor-bearing mouse model was induced by injecting Lewis lung cancer cells into the right anterior leg subcutaneously. Sixty-four C57BL/6 J mice were evenly randomized into eight groups, comprising: a control group; an irradiation group; a CpG ODN 0.15 group; a CpG ODN 0.3 group; a CpG ODN 0.45 group; a CpG 0.15 + irradiation group; a CpG 0.3 + irradiation group; and a CpG 0.45 + irradiation group. Tumor growth, serum tumor necrosis factor-alpha and interleukin-12 concentrations, spleen and thymus exponents, and effect of CpG on the secondary immune response were measured, and apoptosis of tumor cells was investigated using TdT-mediated dUTP nick end labeling (TUNEL) after treatment., Results: Tumor volumes in the treated groups were smaller than in the control group, with those of the CpG 0.45 + irradiation group being the smallest. TUNEL showed that the apoptosis rate in all the active treatment groups was higher than in the control group. CpG ODN apoptosis rate, serum tumor necrosis factor-alpha and interleukin-12 levels, and the spleen and thymus exponent showed greater improvement in the groups receiving combination therapy of CpG ODN and irradiation than the control group or the group receiving irradiation alone. With the increasing concentration of CpG ODN 1826, its effect became more and more significant, meanwhile, inoculation of Lewis lung cancer cells failed in those CpG ODN-cured mice., Conclusion: CpG ODNs dramatically increased the radiosensitivity of Lewis lung cancer and enhanced immune function in mice in a dose-related manner.