NF-?B,LncRNA-uc002jit.1 And PARP1 Form A Feedback Loop That Regulates DNA Damage Repair Induced By Daunorubicin In Acute Myeloid Leukemia Cells

PurposeDNA damage is the common mechanism induced by radiotherapy and chemotherapy in the clinical treatment of cancer.The types of DNA damage are widespread,among them,double-strand break damage is the most deterious,whose repair is mainly achieved by two mechanisms:homologous recombination?HR?and non-homologous end joining?NHEJ?.Abnormal activation and enhancement of DNA repair capacity is an important molecular basis for tumor resistance to DNA-damaging agents,so it is significant to target molecular pathways involved in DNA damage recognition,response and repair.Acute myeloid leukemia?AML?is an invasive malignant tumor,the problems of drug resistance and recurrence are prominent.Sustained activation or high activity of NF-?B was detected in AML cells as well as AML stem and progenitor cells,radiation or DNA-damaging agents can also activate NF-?B specifically,which regulates the transcription of apoptosis and repair related genes,causing radiotherapy or chemotherapy resistance.Poly?ADP-ribose?polymerase 1?PARP1?is a ribozyme closely related to DNA repair and is involved in multiple DNA repair pathways such as BER,HR and NHEJ.PARP1 senses DNA damage and recruits related proteins through PARylation to activate NF-?B.Long noncoding RNAs?LncRNAs?can regulate gene expression at epigenetic,transcription and post-transcription,and they are widely involved in DNA damage repair,cell cycle regulation and other physiological or pathological processes to regulate tumor development.This study aimed to explore the specific molecular regulations of NF-?B,PARP1,and LncRNA-uc002jit.1,and deep theoretical study on NF-?B regulating DNA damage repair,and eventually find new targets and strategies for eradicating AML cells.Methods1 NF-?B and PARP1 form a positive feedback loop to regulate DNA damage repair in acute myeloid leukemia cellsRELA knockdown and overexpression AML cell lines were constructed to verify the effect of RELA on DNA damage repair induced by DNA-damaging agent daunorubicin?DNR?in AML cells.DR-GFP and EJ5-GFP reporter plasmid analysis was used to detect the effect of RELA on HR and NHEJ repair pathways,and the effect of RELA on PARylation was detected by Western blotting.Real-time quantitative PCR and Western blotting were used to detect the effect of RELA on PARP1 mRNA and protein expression.The dual luciferase reporter gene assay was performed to verify that transcription of PARP1 was regulated by RELA.We identified RELA-binding motifs in the PARP1 promoter using the bioinformatic prediction tool JASPAR.We applied chromatin immunoprecipitation and DNA pull down to demonstrate whether RELA binds to the PARP1 promoter in vivo and in vitro.PARP1 knockdown AML cell lines were constructed to verify the effect of PARP1 on NF-?B activation upon DNA damage in AML cells using the secreted luciferase reporter system.2 Double inhibition of NF-?B and PARP1 increased the efficacy of DNR in vitro and in vivoThe secreted luciferase reporter system was used to analyze the effect of the combination of NF-?B inhibitor BMS-345541 and PARP1 inhibitor Olaparib on NF-?B activity in AML cells upon DNA damage.Pathway reporter plasmid analysis and western blotting were used to test the effect of pharmacological inhibition of both NF-?B and PARP1 on DNA repair in AML cells.Flow cytometry,confocal microscopy analysis and the neutral comet assay were used to detect the effect of pharmacological inhibition of both NF-?B and PARP1 on the DNR-induced DNA damage accumulation in AML cells.Annexin-V-FITC and PI double staining was applied to measure the effect of pharmacological inhibition of both NF-?B and PARP1 on the DNR-induced apoptosis in AML cells.MTT assay was conducted to analyze the effect of pharmacological inhibition of both NF-?B and PARP1 on sensitizing AML cells to DNR in vitro.KG1?cell xenograft tumor nude mouse model was constructed to test the effect of pharmacological inhibition of both NF-?B and PARP1 on sensitizing AML cells to DNR in vivo.3 Mutual regulation of RELA/uc002jit.1/PARP1 affected DNA damage repair and cell proliferation in acute myeloid leukemia cellsWe used a high-throughput LncRNA microarray to analyze the differential expression of LncRNA between AML^shCtrl and AML^shRELAhRELA cells upon DNA damage,calculated the Pearson correlation coefficient?PCC?between coding and noncoding by R language,drew CNC?coding-non-coding?co-expression network to look for LncRNAs with positive correlation with RELA and PARP1 mRNA with PCC?0.98.Real-time quantitative PCR was performed to verify the relative expression of uc002jit.1 in the microarray.Luciferase reporter assays were used to identify uc002jit.1as a novel NF-?B target regulated by RELA.RELA-binding motifs in the uc002jit.1promoter was identified by JASPAR.Chromatin immunoprecipitation and DNA pull down assay were applied to demonstrate whether RELA binds to the uc002jit.1promoter in vivo and in vitro.uc002jit.1 knockdown AML cell lines were constructed to verify the effect of uc002jit.1 on the expression and stability of PARP1 mRNA by real-time quantitative PCR.Western Blotting was used to examine the effect of uc002jit.1 on the PARP1 protein expression and PARylation upon DNA damage.Flow cytometry was used to detect the effect of uc002jit.1 on DNA damage repair in AML cells.We also detected the effect of uc002jit.1 on cell proliferation by cell counting,EdU staining and colony formating,then the colony formation assay was used to determine its sensitization effect on DNR in vitro.The uc002jit.1 knockdown AML cell xenograft tumor was constructed to tested for the effect of uc002jit.1 on AML cells proliferation and DNR sensitization in vivo.Results1 NF-?B and PARP1 form a positive feedback loop to regulate DNA damage repair in acute myeloid leukemia cellsRELA regulated the HR and NHEJ repair pathway of DNA damage in AML cells.It binded to the promoter of PARP1 to regulate its transcription and affected the PARylation upon DNA damage repair in AML cells.Conversely,PARP1 also affected the activation of NF-?B upon DNA damage in AML cells,which demonstrated that they formed a positive feedback regulation loop.2 Double inhibition of NF-?B and PARP1 increased the efficacy of DNR in vitro and in vivoDouble inhibition of NF-?B and PAPR1 more effectively than single treatment in inhibiting the repair of DNA damage,increasing the accumulation of DNR-induced DNA damage,promoting DNR-induced apoptosis,inhibiting DNR-induced proliferation in AML cells,and eventually more effectively inhibited the growth of AML cell xenograft tumors and prolonged the survival time of xenograft mice.3 Mutual regulation of RELA/uc002jit.1/PARP1 affected DNA damage repair and cell proliferation in acute myeloid leukemia cellsLncRNA microarray showed that 106 lncRNAs were differentially expressed by at least 2-fold?P<0.05?in the KG1?^shRELA cells.Of these,78 were up-regulated and 28were down-regulated.We obtained 11 lncRNAs positively correlated with RELA and PARP1 mRNA with the PCC?0.98 by coding-noncoding network analysis.Among them,uc002jit.1 was the most significantly down-regulated.The transcription of uc002jit.1 was regulated by RELA,and mainly distributed in the cytoplasm.uc002jit.1affected the stability of PARP1 mRNA,and consequently the expression of PARP1protein and the PARylation in AML cells upon DNA damage,so that affecting DNA damage repair and cell proliferation,sensitivity of AML cells to DNR in vitro and in vivo.ConclusionsRELA regulated the transcription of PARP1 mRNA and LncRNA-uc002jit.1,and then uc002jit.1 affected the stability of PARP1 mRNA at the post-transcription.These three molecules formed a positive feedback loop,which regulated DNA damage repair in multiple pathways.Therefore,if the drug can simultaneously inhibit two important molecules?NF-?B and PARP1?in the loop,the inhibitory effect will be amplified by the loop's positive feedback mechanism,resulting in a more pronounced treatment effect.Further more,we found a completely new LncRNA-uc002jit.1,which has significant biological functions in DNA damage repair,cell proliferation and sensitizing chemotherapeutic drugs in vitro and in vivo.In conclusion,our study provided new targets for AML treatment,and new biomarkers for the prognosis of AML chemotherapy.It also provided new theoretical basis for PARP1 inhibitors used for AML therapy,which would broaden the use of PARP1 inhibitors.