Role And Mechnasim Of TBK1in Radiation-induced Epithelial-mesenchymal Transition Of Alveolar Epithelial Cells

Nowadays, nuclear is playing an essential role in fields such as industry, nation defense,technology, medicine and so on. It not only can bring benefit to human beings, but alsoinevitably take radiation-related dangers for us, which may pollute our environment and moreimportantly challenge human bodies’ healthy. Nuclear technology of our county is developingso rapidly that we possess the strength of nation defense, such as nuclear weapons and nuclearsubmarine, meanwhile, we have built several nuclear power station. In2011, Japan’sfukushima nuclear leak because of an earthquake of magnitude9.0on the Richter scale havecaused a great loss to Japan and at the same time produced panic and scare to our country. Asthe continuing proceed of modern biological technology, radiation therapy has become one ofthe most popular treatments of malignant tumors. Emerging evidence has suggested thatradiotherapy exert its therapeutic effect by killing tumor cells and increasing radiation dosesfor treating malignant cells is followed by inevitable toxic effects for normal tissues. Despitethe advancement of modern radiotherapeutic measures and technology, the toxic effects stillrestrict the widespread use of radiotherapy.Radiation-induced lung fibrosis is one of the common complications of patients who aretreated with radiotherapy and exposed to radiation in nuclear accidents. It contributes to thefailure of radiotherapy in malignant tumor patients and the death of patients who suffer fromthe late stage of acute radiation diseases. The mechanism of lung fibrosis remains unclear andthe prevention and treatment of lung fibrosis is urgent to clinical doctors, so a key element inthe pathogenesis of lung fibrosis, which acts as a target at investigating the relationship of IRand RILF, will supply new opinions for formulating clinical radiotherapy strategy, enhancingradiotherapy effect and reducing the side-effects.Lung fibrosis is characterized by the injury of alveolar epithelial cells and theaccumulation of fibroblasts or myofibroblasts, which is companied by the deposition ofcollagen and extracellular matrix (ECM) that consequently interfere the repairmen of normallung tissues and result in lung fibrosis. As the vital role of myofibroblasts in the pathogenesis,the origin of it has become the focus of intensive research. Evidence has indicated that underthe stimulation of injury factors the epithelial cells can loss their epithelial character andacquire the mesenchyal character. This biological process is called epithelial-mesenchymaltransition (EMT). EMT is demonstrated in the animal model and patients’ tissues of idiopathicpulmonary fibrosis (IPF). EMT, which is mediated by some factors, is related to the activation of specific transcription factors, the increase of specific membrane proteins, the reconstructionof cellular skeleton proteins, the degeneration of matrix metalloproteinases and the change ofspecific microRNAs. Radiation is an important inducer of EMT. A number of distinctmolecular processes are engaged in order to initiate or inhibit an EMT, including miR-200c,and TBK1is the direct target of miR-200c. Thus, investigating the relationship of TBK1onEMT induced by radiation and the signaling pathway of TBK1in this biological process willoffer some essential proof for us to clarify the relationship between IR and RILF.In the present study, we choose alveolar epithelial type Ⅱepithelial cells as the object.The effect of IR on EMT will be estimated by some EMT-related specific markers after thecells were exposed toγ-rays. On account of the changeable expression of TBK1in the cellsafter radiation, we next use the siTBK1to investigate the role of TBK1in this pathogenesisand discuss its possible downstream pathways on radiation-induced EMT.Contents of study:1、The role of radiation on EMT: The human alveolar type Ⅱepithelial cell line A549andrat alveolar type Ⅱepithelial cell line RLE-6TN were irradiated with a variety dose of γ-rays and detect the EMT markers at different time points, in order to establish the cell modelof IR-induced EMT;2、The role of siTBK1on radiation-induced EMT:①Transfer siTBK1to cells by lipo2000and clear about it;②Using siTBK1to knockdown TBK1levels in cells and observe thealteration of EMT markers;3、The mechanism of TBK1on radiation-induced EMT:①Using siTBK1to knockdownTBK1levels in cells and detect the change of EMT-related transcription factors;②Themechansim of TBK1on radiation-induced EMT.Methods:1、The human alveolar type Ⅱepithelial cell line A549and rat alveolar type Ⅱepithelialcell line RLE-6TN were irradiated with a variety dose of γ-rays and detect the EMTmarkers at different time points, then the changes of EMT markers were detected by westernblot;2、Western blot was used to detect the expression of TBK1after radiation;3、Using lipo2000to transfer siTBK1into cells, and detect the effect of siTBK1viaReal-time PCR and western blot. 4、Using siTBK1to knockdown TBK1levels in cells and detect the change of EMTmarkers by western blot and immunofluorescence;5、Using siTBK1to knockdown TBK1levels in cells and detect the change of EMT-relatedtranscription factors; And rescue was used to explicit the role of transcription factor,ZEB1;6、Using siTBK1to knockdown TBK1levels in cells and using real-time PCR toinvestigate the effect of IR on the expression of TGF-βin cells. Then the role of TGF-β/Smad signal pathways in IR-induced EMT was discussed in the group of siTBK1and thecontrol;7、The role of NF-κB signaling pathway was tested in the group of siTBK1and the controlafter radiation;8、The role of GSK-3βwas detected in the group of siTBK1and the control after radiation;then the change of EMT makers and ZEB1was detected using the specific inhibitor of GSK-3β, SB216763.9、Statistical analysis: we conducted a Student’s t-test and analysis of variance (ANOVA).And statistical significance was declared at the P less than0.05.Results:1、A549cells were irradiated with a single dose of2,4,6, and8Gy γ-rays. At the proteinlevel, radiation reduced expression of the epithelial marker E-cadherin and enhanced theexpression of the mesenchymal marker vimentin in a time-and dose-dependent mannercompared with non-irradiated control levels.2、After the irradiation of8Gy γ-rays, the expression of TBK1was increased in atime-dependent manner.3、Using siTBK1to knockdown the TBK1levels via lipo2000, the expression of TBK1wasdecreased obviously.4、Using TBK1siRNAs to knockdown TBK1levels in cells, the normally observedinduction of vimentin caused by radiation treatment was attenuated and the decrease in basalE-cadherin levels was prevented; Taken together, our data suggest that TBK1inhibitionattenuates radiation-induced responses in cells.5、The mRNA level of ZEB1was induced at3h after radiation and silencing TBK1 attenuated the change of ZEB1. Meanwhile, radiation caused a time-dependent increase inZEB1protein level, however, TBK1knockdown attenuated the increasing expression ofZEB1protein induced by radiation. Overexpression of ZEB1overrode the role of TBK1inhibition on radiation-induced EMT. Based on the result, we viewed TBK1-regulatedradiation-induced EMT via repression of ZEB1.6、The TGF-β contents were enhanced in A549after irradiation, but silencing TBK1didnot affect TGF-β. The expression of p-Smad3in A549was increased after irradiation,whereas the level of smad3remained unchanged. Whereas attenuation of TBK1did notdecrease the radiation-induced expression of p-Smad3, indicating the minimal requirement ofsmad signaling in TBK1effect.7、Radiation could degrade as well as phosphorylate IkB-a within3hours, indicating thatradiation may or can activate NF-kB pathway. Inhibition of TBK1by using siRNA did notaffect the protein level of IkB-a and p-IkB-a induced by radiation. These findings indicate thatactivation of the NF-kB signaling pathway is not required for TBK1inhibition onradiation-induced EMT.8、Inhibition of TBK1by using siRNAs resulted in a significant decrease in phosphorylatedGSK-3β in cells, which indicated that TBK1inhibition could overrode the effect thatradiation inactivated GSK-3β. Stimulation with SB216763attenuated the expression ofbiomarker specific for EMT and decreased the expression of transcription factor ZEB1compared with only inhibition of TBK1by using siRNAs. These findings imply that GSK-3βsignaling is capable of producing a powerful inhibitory signal for radiation-induced EMT,suggesting that phosphorylation of GSK-3β by TBK1inhibition is a plausible explanationfor its anti-EMT effects.Discussions and Conclusions:RILF is one of the common complications of patients who are treated with radiotherapyand exposed to radiation in nuclear accidents. It contributes to the failure of radiotherapy inmalignant tumor patients and the death of patients who suffer from the late stage of acuteradiation diseases. RILF has become the focus of intensive study and the main problems ofradiotherapy that urge to resolve in domestic and overseas in radiation biology areas.Nowadays, a variety of researches have emphasized the critical role of EMT in lung fibrosis,and radiation is one of the important inducer of EMT. Our laboratory has identified that TBK1is the direct target of miR-200c, which plays a regulatory role on EMT, and have an oncogene-like role. Based on these, we expect to demonstrate the role of TBK1onradiation-induced EMT, in order to provide some important evidence to clarify the mechanismof radiation-induced lung fibrosis. In this study, we tested whether TBK1has a role inradiation-induced EMT and identified associated potential mechanisms. Human and ratalveolar type II epithelial cells were irradiated with60Co γ-rays. Western blotting revealed atime-and dose-dependent decrease in E-cadherin with a concomitant increase in vimentinafterradiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. TBK1siRNA significantly inhibited radiation induced suppression of the epithelial markerE-cadherin and up regulation of the mesenchymal marker vimentin.Furthermore, radiation-induced EMT attenuated by TBK1depletion was partiallydependent on transcriptional factor ZEB1expression. Finally, we found GSK-3βis involvedin regulation of radiation-induced EMT by TBK1. Thus, our findings reveal that TBK1signaling regulates radiation-induced EMT by controlling GSK-3βphosphorylation andZEB1expression. TBK1may therefore constitute a useful target for treatment ofradiotherapy-induced fibrosis diseases.Conclusion:IR can induce the process of EMT, and TBK1signaling regulates radiation-induced EMTby controlling GSK-3β phosphorylation and ZEB1expression. TBK1may thereforeconstitute a useful target for treatment of radiotherapy-induced fibrosis diseases.