| Objective:Squamous cell carcinoma（SCC）is the second most common malignancy of the skin.Worldwide,about 700,000 SCCs are diagnosed each year.Although the degree of malignancy of SCC is low,lymphatic and/or other organ metastases occur in approximately 14%of cases,and eventually death occurs in approximately 40%of patients with metastases.In recent decades,the incidence of SCC has been increasing,which has caused a heavy economic burden to patients and the society.Therefore,clarifying the potential pathogenesis of SCC and clarifying the relevant therapeutic targets are urgent scientific issues to be solved in this field.The basic leucine-zipper（BZIP）family is one of the core transcription factors that are widely present in vivo and responsible for regulating oxidative stress in vivo.Nuclear factor erythroid 2-related factor 1（NRF1）,a major member of the b ZIP family,plays an important role in embryonic development,regulation of inflammatory response and maintenance of proteasome homeostasis.At the same time,there are also literatures showing that NRF1is also involved in the occurrence and development of cancer.However,the association between NRF1 and SCC has not been reported.Therefore,in this study,we intend to use keratinocytes NRF1 specific knockout mice to clarify the role of NRF1 in the development and progression of chemically carcinogenic SCC through in vivo experiments.At the same time,we combined with lentivirus stably transfected NRF1gene silenced Ha Ca T cell lines to explore the potential mechanism of NRF1 antitumor effect.So as to clarify the pathogenesis of SCC to provide a new theoretical basis.Methods:1.Optimization of SCC model in mice induced by chemical carcinogensWild type mice with C57BL/6J genetic background were selected.After anesthesia,the back skin was depilated（2 cm×2 cm）.After 48 hours,7,12-Dimethylbenzo[a]anthracene（DMBA）was applied.After one week,Phorbol 12-myristate 13-acetate（TPA）was applied twice a week on the same depilation site.The smear concentration of DMBA and TPA was divided into the following three groups,namely 97.5 nmol DMBA+6.5 nmol TPA;97.5 nmol DMBA+16 nmol TPA;200 nmol DMBA+16 nmol TPA and solvent control（Acetone）.The skin tumor formation of mice in each group was monitored regularly weekly after TPA smear,so as to screen and establish the optimal DMBA/TPA smear induced SCC experimental scheme.2.The role of NRF1 in DMBA/TPA induced SCC in keratinocytesKeratinocyte specific knockout（Nrf1（K14）-KO）mice were constructed by crossing NRF1-Flox and K14-Cre+/-transgenic mice under the genetic background of C57BL/6J,and their lute-born control mice（Nrf1-Flox）mice were obtained.According to the optimal DMBA/TPA smear scheme obtained in Study 1,the above two genotypes of mice were respectively smeared with DMBA/TPA or Acetone,with 5-12 mice in each group.The body weight of mice in each treatment group was measured weekly,the tumor load（tumor occurrence time,tumor size,tumor number）was recorded,and the end point（40 weeks after TPA treatment）was observed.Serum and skin tissue samples were collected.3.Effect of Nrf1 deficiency in keratinocytes on DMBA smear induced DNA damageEight-week-old female mice with genotypes of Nrf1-Flox and Nrf1（K14）-KO born in the same lair were selected and given back skin（2 cm×2 cm）Acetone or DMBA smear,with 6 mice in each group.The back skin of mice was collected at 0 h and 24 h after treatment,and the expression of H2AX phosphorylated proteinγ-H2AX in the epidermis was determined by immunohistochemical method.The number of positive cells in the epidermal layer of mice was measured by Image J for statistical analysis.4.NRF1 gene silencing plays a role in DNA damage repair in UVB induced Ha Ca T cells2 J UVB irradiation at 0,12,24,36,48 h after Scramble and NRF1-KD,respectively.Cell proliferation was detected by full field cytometry.He Ca T cells of Scramble and NRF1-KD were irradiated with 2 J UVB for 6 h,respectively.Intracellular DNA damage indexγ-H2AX was detected by immunofluorescence staining.2 J UVB treatment of Scramble and NRF1-KD Ha Cat cells,respectively.Cells were collected before treatment（vehicle）and 0 h,5 min,2 h,6 h and 24 h after treatment,respectively.The protein levels of p-ATR and p-CHK1 were detected by Western blot.5.Statistical analysisAll data in this study were analyzed with Graph Pad Prism 8 software.Results:1.SCC model induced by DMBA/TPA was successfully constructedAccording to the tumor formation of mice in different experimental regimens,Wefound that skin tumors in mice of 97.5 nmol DMBA+16 nmol TPA group formed earlier and had more numbers,so this smear scheme was used as the final experimental scheme of this experiment.2.Keratinocyte specific Nrf1 deficiency can affect the occurrence and development of DMBA/TPA induced skin tumors in miceThere was no significant difference in the body weight of mice among all groups,suggesting that NRF1 deficiency had no effect on the body weight of mice.After TPA and Acetone were smeared,the tumor load of each group of mice was dynamically recorded continuously,and the results showed that,The back skin of mice treated with Acetone has no change in appearance.However,in the DMBA/TPA treated mice,the number of skin tumors gradually increases with the extension of TPA application time.he tumor incidence of Nrf1（K14）-KO mice was higher than that of NRF1-Flox mice from 13weeks of TPA treatment.The difference was statistically significant（p<0.05）。In the comparison of tumor diameter,we selected two time points 20 weeks and 40 weeks after TPA application for comparison.The results showed that at 20 weeks,the number of tumors in Nrf1（K）-KO mice was higher than that in Nrf1-Flox mice,no matter in the diameter range of 1mm-2 mm,2mm-5 mm or larger than 5 mm,but only the difference between the two groups was statistically significant in the diameter range of 2mm-5mm.At 40 weeks,the comparison results within the diameter range of the above three groups showed that the number of tumors in Nrf1（K14）-KO mice was higher than that in Nrf1-Flox mice,and the difference was statistically significant（p<0.05）.3.Keratinocyte specific Nrf1 deficiency can affect DMBA daub induced skin DNA damage in miceAfter DMBA treatment for 0 h,there was no significant difference between Nrf1（K14）-KO and Nrf1-Flox cells inγ-H2AX positive staining,After DMBA treatment for24 h,γ-H2AX positive cells were found in both groups,and the number ofγ-H2AX positive cells in the epidermis of Nrf1（K14）-KO mice was significantly higher than that of Nrf1-Flox mice.The difference was statistically significant（p<0.05）.4.NRF1 gene silencing can affect DNA damage induced by UVB irradiation of Ha Ca T cellsThe expression ofγ-H2AX and 53BP1 in Hacat cells was measured byimmunofluorescence method 6 hours after irradiation of Hacat cells with 5 J UVB.The number and brightness ofγ-H2AX and 53BP1 in NRF1-KD group were higher than that of Scramble group.5.NRF1 gene silencing can affect the expression of protein molecules related to ATR/CHK1 signaling pathway during DNA damage repairAfter 2J UVB irradiation of Ha Ca T cells,the expression levels of p-ATR and ATRprotein in NRF1-KD group were not significantly changed,but the protein expression levels of p-CHK1,CHK1,p-cdc2 and cdc2 were all lower than those in the control group.Conclusion:1.Nrf1 deficiency aggravated the occurrence of skin tumors induced by DMBA/TPA in mice.2.The loss of Nrf1 aggravated the DNA damage caused by a single DMBA treatment of mouse dorsal skin.3.NRF1 silencing inhibited UVB irradiation-induced upregulation of p-CHK1protein levels... |
PDF Full Text Request