The Effect And Mechanism Of Long Non-conding RNA PANDAR On Non-small Cell Lung Cancer Sensitivity To Radiation And Chemotherapy

BackgroundWith non-small cell lung cancer(NSCLC)accounting for roughly 85%,lung cancer is one of the most commonly diagnosed cancers worldwide.Despite advances in targeted therapies and immune checkpoint inhibitors over the past decades,NSCLC remains an incurable disease for most patients.Platinum-based chemotherapies and radiation,both inducing DNA damage and cytotoxicity,are widely used as anticancer therapies.Nevertheless,cancer cells tend to be resistant to both chemotherapy and radiation,thus resulting in treatment failure or recurrence.DNA damage and replication stress induced from chemotherapies and radiation will trigger the activation of DNA damage repair pathways and checkpoints,the latter providing the time necessary for DNA repair.The process is frequently described as DNA damage response(DDR),which is also closely linked with the senescence or apoptotic or other machineries to enable the elimination of cells with unrepaired DNA damage and ultimately remain body homeostasis.DDR defects contribute to genomic instability and promote the evolution of cancer cells through the accumulation of genetic mutations and rearrangements.However,thanks to those defects,cancer cells are vulnerable to interventions of chemotherapies and radiation while normal cells fight against them.Plenty of studies have shown that tumor cells with abnormally enhanced DNA damage repair ability can effectively avoid cell death caused by DNA damage and further proliferate,thus playing a role in resistance to radiation and chemotherapies.A variety of lnc RNAs have regulate such resistance through impacting DNA damage repair,apoptosis and tumor stem cell activity.With RNA immunoprecipitation sequencing,our previous study has shown that LncRNA PANDAR is capable of binding to ATR,which is a key molecule for DNA damage repair.After reviewing related literature,we learn that it is closely relevant to DNA damage.Contents and Methods1.Effect of LncRNA PANDAR on NSCLC Cells Sensitivity to Radiation and ChemotherapyNSCLC cell lines A549 and H1299 were used to construct PANDAR knockdown and over-expression stably transformed cells,then we treated them with cisplatin and γ-ray.We performed experiments of CCK-8、colony formation assay、cell cycle、APC/PI double staining flow cytometry,exploring the effect of LncRNA PANDAR on NSCLC Sensitivity to Radiation and Chemotherapy.2.Regulatory Mechanism of LncRNA PANDAR on NSCLC Cells Sensitivity to Radiation and ChemotherapyCell lines of PANDAR knockdown,overexpression and control were respectively treated with cisplatin,γ-ray and control.We evaluated the extent of DNA breaks using comet assay and performed the immunofluorescence staining against γH2AX-indicator of DNA damage.In addition,we explored the role of PANDAR in DDR pathways through western blot.3.Effect of LncRNA PANDAR on Sensitivity to Radiation and Chemotherapy in NSCLC Nude Mice ModelWe established nude mouse subcutaneous xenograft models using A549 Knockdown cells and A549 wild cells as control.Mice with similar baseline were randomly selected and divided into different groups,treated with cisplatin or irradiation.Then,tissues were taken for immunohistochemistry and detecting Ki67 for tumor proliferation,TUNEL for apoptosis,γH2AX for DNA damage.Through the above experiments,we explore the effect of PANDAR on sensitivity to radiation and chemotherapy in vivo.Results1.LncRNA PANDAR Negatively Regulated Sensitivity to Radiation and Chemotherapy in NSCLC CellsIn cell experiments,PANDAR overexpression can increase the resistance of NSCLC to radiation and chemotherapy.The CCK8 results showed that cell viability was significantly increased in the overexpression group after radiation and cisplatin treatments.The overexpression group also invited more colonies and apoptosis,and cell cycle arrest was more severe so that providing the time necessary for DNA repair.Contrary to PANDAR overexpression,the trends were reversed in PANDAR knockdown group.2.LncRNA PANDAR Promoted Repair of Radiation and Cisplatin-induced DNA Damage Through ATR/CHK1 in NSCLC CellsComet assay results showed that more damages were observed in PANDAR knockdown group cells treated with radiation,whereas the opposite effect was observed in overexpression group.Furthermore,we performed the immunofluorescence staining against γH2AX-indicator of DNA damage.The PANDAR knockdown group of both A549 and H1299 cells led to decreases of γH2AX foci following cisplatin and radiation treatment,while the amounts in the PANDAR overexpression group were decreased.These results indicated that PADDAR has an impact on sensitivity to radiation and chemotherapy in NSCLC through regulating DNA damage repair.Proteins related to DDR pathway were detected by western blot,it was found that the phosphorylation levels of ATR and CHK1 in PANDAR knockdown groups treated with radiation and cisplatin were inhibited to a certain extent,indicating that PANDAR can promote the repair of DNA damage through ATR / CHK1 pathway,so as to increase the resistance to radiation and chemotherapy.3.LncRNA PANDAR Knockdown Enhanced Sensitivity to Radiation and Chemotherapy in NSCLC Nude Mice ModelMice-borne tumors of PANDAR knockdown had better response(smaller tumor volume)than control group after radiation or cisplatin treatment for 3 weeks(P < 0.01).IHC staining of xenograft tumors showed that tumors derived from PANDAR knockdown cells exhibited stronger γH2AX staining than control group after treatment of radiation or cisplatin.In comparison with control group,we observed significantly higher TUNEL and Ki67 staining in cisplatin or radiation-treated PANDAR knockdown group,which respectively reflected the apoptosis level and proliferation ability.ConclusionIn this study,with experiments in cells and animal model,we found that PANDAR knockdown promoted sensitivity to radiation and cisplatin-induced chemotherapy in NSCLC,which is affected by regulating DNA damage repair.We also found that PANDAR knockdown inhibited the phosphorylation of ATR / CHK1 pathway,thus resulting in better response to radiation and cisplatin-induced chemotherapy.This study provided novel insights into the mechanism of PANDAR promoting DNA damage cell survival.Promising PANDAR-targeting therapeutic in sensitizing NSCLC to DNA damaging drugs may ultimately enter clinical trials,which is of great significance to improve the prognosis of NSCLC patients.