A Study Of S100A16 Nuclear Translocation In Lung Cancer Cells Under Oxidative Stress To Promote Ribosome Production

Objective:Lung cancer is the malignant tumor with the highest morbidity and mortality in China.lung cancer can be classified into non-small cell lung cancer（NSCLC）and small cell lung cancer（SCLC）according to pathological classification.the occurrence of lung cancer is associated with smoking,occupational exposure,air pollution,and pulmonary infectious diseases,but the exact mechanism is unclear.Therefore,understanding the pathogenesis of lung cancer is of great significance for the early diagnosis of the disease.In the early stage of lung cancer,oxidative stress injury is involved in the development of lung cancer and induces carcinogenesis of normal lung tissue cells.In the advanced stage of lung cancer,lung cancer cells show strong ability of anti-oxidant stress,and then anti-apoptosis and resistance to chemotherapeutic drugs.Therefore,it is helpful to understand the malignant process of lung cancer and the mechanism of chemoradiotherapy resistance.Previous work found that lung cancer cells expressed high S100 family protein——S100A16,.and associated with the viability of lung cancer cells after metastasis into the brain.S100 family proteins are stress proteins,which have been reported to be involved in acute and chronic inflammatory stress reactions,and are closely related to the occurrence and development of many tumors.This study took S100A16 as the research object to explore the transformation of intracellular S100A16 expression and intracellular protein localization in lung cancer cells under oxidative stress conditions,and the effect of this transformation on the biological phenotype of lung cancer cells.And we try to find out how S100A16 mediated protective mechanism can help lung cancer cells to resist oxidative stress injury and possible mechanism,which has certain research value for elucidating the mechanism of lung cancer occurrence and development.Method:1.Western blot detection of S100A16 protein expression in different pathological types of tumor cells.2.To give hydrogen peroxide（H₂O₂）stimulation to analyze the changes of lung cancer cell NCI-H446 and A549 intracellular expression under oxidative stress.3.Using cell component separation kit,lung cancer cells（NCI-H446 and A549 cells）were separated by different cell components（cytoplasm and nucleus）.The expression of S100A16 in different components was analyzed by western blot;immunofluorescence results laser confocal microscope was used to observe the localization of S100A16 in cells under oxidative stress conditions;and dual immunofluorescence staining was used to observe the co-location of S100A16 with nuclear marker molecules.4.Prokaryotic expression system of GST-S100A16 fusion protein was constructed,GST-S100A16 fusion protein was purified by affinity chromatography,GST Pull Down binding immunoprecipitation method（Co-IP）was used to capture the protein that could bind to S100A16,and differential electrophoresis bands were selected by polyacrylamide gel electrophoresis to obtain the P95.of candidate proteins which could interact with S100A16 under oxidative stress and have nuclear translocation5.GST Pull Down and CO-IP techniques were used to verify the interaction between S100A16 and P95 proteins,and immunofluorescence assay was used to verify the interaction between them under oxidative stress.6.We designed experiments to demonstrate the interaction between S100A16 protein and nucleolar protein P95 oxidative stimulation.first,the GST Pull Down and CO-IP techniques were used to verify the interaction of the two proteins;next,the immunofluorescence experiment was used to verify that the two proteins interacted under the condition of oxidative stimulation,and after silencing the P95 protein,the oxidative stimulation was given to observe whether the two proteins combined.This shows that S100A16 protein can enter the nucleus under oxidative stimulation,which is P95 with nucleolar protein.7.Real-time fluorescence quantitative PCR and western ablot analysis S100A16 the effect on ribosome expression after entering the nucleus.real-time fluorescence quantitative PCR analysis 47 expression change.Result:1.Western blot results showed that S100A16 were significantly expressed in lung adenocarcinoma A549 and small cell lung cancer cell NCI-H446 and NCI-H1688,but hardly in other tumor cells（large cell lung cancer NCI-H460,small cell lung cancer,and ovarian cancer cell CAVO3）.to this end,this study selected A549 and NCI-H446 as further research subjects.2.H₂O₂（50 um）was treated with H₂O₂（HBMEC）for 2 h in the control of brain microvascular cells.western blot results showed that the expression level of the S100A16between the NCI-H446 cells and the A549 cells given the treatment was significantly increased,but there was no significant change in the expression in the normal.3.The NCI-H446 and A549 components of lung cancer cells before and after H₂O₂stimulation were isolated.The results showed that H2O2 stimulation could reduce the content of S100A16 in cytoplasm and increase the content content in the nucleus,which suggested that S100A16 had a change of position in the cell,which could enter into the nucleus by cytoplasm.H2O2 can induce S100A16 to translocate from the cytoplasm into the nucleus;and how S100A16 is clearly localized in the nucleolar region.4.The prokaryotic expression system of GST-S100A16 fusion protein was successfully constructed,and the GST-S100A16 fusion protein was obtained by affinity chromatography,GST Pull Down the protein that could bind to S100A16 under H₂O₂action was found in lung cancer cells.A candidate molecular-P95 was obtained by immunoprecipitation（Co-IP）to capture the binding protein in lung cancer cells after treatment.5.The interaction between S100A16 and P95 in lung cancer cells under H2O2 action was analyzed by immunoprecipitation technique.The results showed that in the presence of H₂O₂ action conditions,the direct interaction between S100A16 and P95.6.To explore the possible role in nuclear S100A16,we analyzed the main functions of the nucleolus,rRNA and and ribosome synthesis condition,real time PCR analysis of rRNA precursor pre-47s content,prompt S100A16 after entering the nucleus to induce transcriptional body rDNA pre-47 s content,these preliminary results suggest S100A16after entering the nucleus in nucleolus,may participate in the nucleolus of ribosome synthesis process,The mechanism needs to be further explored.Conclusion:1.Oxidative stimulation can increase S100A16 protein in lung cancer cells.2.Conditions of oxidative stimulation,the binding P95 S100A16 proteins to nucleolar proteins helps to enter and locate nucleoli.3.Binding of S100A16 protein to P95 protein promotes ribosome regeneration under oxidative stimulation.