The Role And Mechanism Of NRF1-PARP1 Interaction In Regulating DNA Damage Response Against The Development Of Urethane-induced Lung Adenocarcinoma

Objective:Lung cancer is a major global public health challenge due to its high incidence and mortality rates,which pose a serious threat to human health and life.Genetic and environmental factors can induce the development of cancer.Chemicals,as the most common carcinogenic factors in the environment,are frequently to see to activate the core transcription factors involved in the regulation of environmental stress response to participate in the development and progression of cancer.Nuclear factor erythroid 2-related factor 2（NRF2/NFE2L2）plays an important protective role in chemical and radiation-induced carcinogenesis models.However,in cancer cells,the activation of NRF2 has been found to promote progression,metastasis,and inhibit chemosensitivity.Nuclear factor erythroid 2-related factor 1（NRF1/NFE2L1）can also regulates a series of intracellular response in cells and participate in the regulation.However,Nrf1 global knockout mice do not survive.There were a few studies related to the physiological functions and pathological effects of NRF1,especially in cancer.Urethane（Ure）is a chemical widely found in fermented foods and alcoholic beverages.It is also a commonly used modeling drug in lung cancer research.The main target cells of its activated metabolites are alveolar epithelial type II cells（ATII）.DNA damage caused by chemicals is one of the major mechanisms of chemical carcinogenesis.Therefore,in this study applied clinical molecular epidemiological and cancer database to investigate the expression of different isoforms of NRF1 in lung adenocarcinoma.At the same time,ATII cell-specific knockout Nrf1 mice were used to establish Ure-induced lung adenocarcinoma mice model and analyze the role of NRF1 in the development of lung adenocarcinoma.The molecular mechanism of NRF1 in chemical carcinogenesis was deeply analyzed from the perspective of DNA damage repair combined with in vitro and in vivo models.This provides new evidence for intervention strategies and treatment methods for carcinogenic chemicals.Methods:1.Determining the expression of NRF1 isoforms in lung adenocarcinoma:GEPIA2,c Bio Portal and TIMER2.0 were used to analyze the NRF1 m RNA expression,NRF1genetic variation,NRF1 expression correlation with prognosis and immune cell infiltration simultaneously.The m RNA of each isoform of NRF1 in human lung adenocarcinoma and normal tissues were detected by RT-q PCR.Detecting the protein level of NRF1 protein in lung adenocarcinoma and normal tissues by Western blot,IC-MS/MS and IHC.Constructing Ure-induced lung adenocarcinoma mouse model.RT-q PCR,Western blot,RNAscope and IHC were used to detect NRF1 expression.Analysis of the similarities and differences in NRF1 expression in mice and human lung adenocarcinoma.2.Analysis of the role of NRF1 in Ure-induced lung adenocarcinoma in ATII cells:Using Nrf1（ATII）-KO mice analysis of mouse basal phenotypes.To construct Ure-induced lung adenocarcinoma mouse model.Record the body weight of mice,organ weight,number of pulmonary surface tumors,diameter of pulmonary surface tumors,HE and immunohistochemical staining for tumor burden assessment.Establish a Ure acute treatment model.γ-H2AX and 53BP1 were detected by immunohistochemistry to identify DNA damage.Analysis of the role and potential mechanisms of NRF1 in Ure-induced lung adenocarcinoma.3.The role andmechanism of NRF1 in DNA damage repair:Cell models with Nrf1silencing and/or overexpression were constructed to build CPT,IR or UV-induced DNA damage models.Assessment of DNA damage by Western blot,cell proliferation,micronucleus,comet assay andγ-H2AX immunofluorescence staining.Application of flow cytometry to detect ROS levels before and after NAC intervention.Using Nrf2（ATII）-KO mice analysis of mouse basal phenotypes.To construct Ure-induced lung adenocarcinoma mouse model.Analysis of the role of NRF2 in Ure-induced lung adenocarcinoma in ATII cells.The expression of DNA damage repair proteins PARP1,PAR and XRCC1 detect by Western blot method.the function of PARP1 in recognizing DNA damage detect by Laser micro-irradiation assay.Analysis of NRF1-PARP1 protein interaction using immunofluorescence staining,database prediction and CO-IP.Western blot assay for PARP1 expression after CHX,proteasome inhibitor intervention.CO-IP analysis of PARP1 protein ubiquitination levels.Results:1.Based on the TCGA database,NRF1-L was mainly expressed in human and mouse lung tissues,while the expression of NRF1-S was extremely low.Compared with the normal tissues,NRF1-L expression was significantly decreased in human or mouse lung lung adenocarcinoma tissues（P<0.05）.Comparison of protein band positions of NRF1 isoforms by overexpression and IC-MS/MS sequencing clearly showed that NRF1-742 was significantly decreased in lung adenocarcinoma tissues（P<0.05）.Immunohistochemical staining of human and mouse lung pathology slices and detection of NRF1 protein in lung adenocarcinoma cells and normal lung cells revealed a significant decrease of NRF1 in lung adenocarcinoma tissues（P<0.05）.Based on the TCGA database,the mutation frequency of NRF1 in lung adenocarcinoma tissues was very low.NRF1 was weakly correlated with immune cell infiltration in lung adenocarcinoma tissues.And there was no correlation between NRF1 and the prognosis of lung adenocarcinoma patients.2.Successful breeding of Nrf1（ATII）-KO mice by crossing SPC-rt TA^+/-and Teto-Cre^+/-mice with Nrf1-Flox mice induced by doxycycline given at the age of 6 weeks.HE,immunohistochemistry and lung function assays showed no significant differences in lung tissue structure and lung function indices in Nrf1（ATII）-KO mice.The lung tissue weight,number of lung surface tumors,and tumor volume of Ure-treated Nrf1（ATII）-KO mice were significantly higher than Nrf1-Flox compartment（P<0.05）.HE and immunohistochemical staining showed that the area of lung tumor,PCNA,F4/80,53BP1andγ-H2AX of lung tissue was significantly more in Ure-treated Nrf1（ATII）-KO mice than Nrf1-Flox compartment（P<0.05）.γ-H2AX and 53BP1 were significantly more in acute Ure-treated Nrf1（ATII）-KO mice lung than Nrf1-Flox compartment（P<0.05）.3.After CPT,IR or UV treatment,the expression of NRF1 andγ-H2AX increased in a dose-dependent manner,and Nrf1 silencing（sh Nrf1）inhibited the expression of NRF1andγ-H2AX proteins（P<0.05）.In addition,cell proliferation rate was significantly decreased and micronucleus number,comet tail length andγ-H2AX nuclear aggregation were significantly increased in the sh Nrf1 cells（P<0.05）.In addition,cell proliferation rate was significantly decreased and micronucleus number,comet tail length andγ-H2AX nuclear aggregation were significantly increased in CPT-treated sh Nrf1 group（P<0.05）.Compared with the sh NC group,ROS levels were significantly higher in sh Nrf1after CPT treatment（P<0.05）.After NAC intervention,compared with the sh NC group,the cells in the sh Nrf1 group still had an increased nuclear aggregation ofγ-H2AX after NAC intervention.There was no significant difference in lung tumor burden in Ure-treated Nrf2（ATII）-KO mice.PARP1,PAR and XRCC1 protein levels were significantly decreased in CPT-treated sh Nrf1 MLE-12 cells than the sh NC group（P<0.05）.Overexpression of Nrf1-L can rescue PARP1 expression.There were similar results in NRF1 silenced Ha Ca T cells.The laser microscopy irradiation experiment found that compared with the sh NC,sh Nrf1 cells inhibited the recruitment of PARP1-GFP to the sites of damage.There was no significant difference in Parp1 m RNA levels between sh NC and sh Nrf1 cells.According to immunofluorescence,molecular docking database prediction and CO-IP,NRF1 directly interacts with PARP1.Compared with sh NC,the degradation rate of PARP1 protein expression in CHX-treated sh Nrf1 cells was significantly faster（P<0.05）.Proteasome inhibitors can rescue the expression of PARP1in CPT-treated sh Nrf1 group.CO-IP found that Nrf1 silencing led to an increase in PARP1 ubiquitination level,while Nrf1-L overexpression could inhibit PARP1ubiquitination level.Conclusion:In DNA damage induced by exogenous mutagens,NRF1 plays a protective role in exogenous mutagens-induced cancer by interacting with PARP1 in the nucleus and inhibiting PARP1 ubiquitination modifications to stabilize its protein expression and DNA damage recognition function.This provides new clues for elucidating the carcinogenic mechanism of exogenous mutagens and provides new strategies for the treatment of exogenous mutagenic carcinogenic interventions.