NAP1L1 Interacts With Hepatoma-derived Growth Factor To Recruit C-JUN Inducing Breast Cancer Growth

1.Background and Purpose:Breast cancer is the top cancer in women in the world.However,its pathogenesis is still to be determined.In this study,Nucleosome Assembly Protein 1 Like 1(NAP1L1)protein was upregulated based on the Clinical Proteomic Tumor Analysis Consortium(CPTAC)database.Consistent with the prediction,immunohistochemistry staining showed that NAP1L1 protein expression was significantly increased in breast cancer tissues.Its elevated expression was an unfavorable factor for breast cancer clinical progression and poor prognosis.Knocking down NAP1L1 reduced the cell cycle progression and growth in vivo and in vitro via repressing cell cycle signal in breast cancer.Furthermore,the molecular basis of NAP1L1-induced cell cycle signal was extensively studied.NAP1L1 interacted with the hepatoma-derived growth factor(HDGF),a potentially oncogenic factor for tumors,and the latter subsequently recruited the key oncogenic transcription factor C-JUN,which finally induced the expression of cell cycle promoter CCND1 and thus the cell growth of breast cancer.This study shows that NAP1L1 functions as a potential oncogene via interacting with HDGF to recruit C-JUN in breast cancer.2.Research contents and methods:2.1 Bioinformatics analysisThe differential expression of NAP1L1 in breast cancer was found in database analysis.HDGF may interact with C-JUN was found in the BioGrid.2.2 Overexpression of NAP1L1 promotes the proliferation of breast cancer cells2.2.1 Ten breast cancer tissues and corresponding paracancerous tissues were selected,Immunohistochemistry showed that the expression of NAP1L1 was high in breast cancer tissues and low in paracancerous tissues.Further immunohistochemistry using tissue microarray(TMA)of 97 human breast tissue samples confirmed that overexpression of NAP1L1 was a detrimental factor in reducing overall survival in breast cancer patients.Multivariate Cox regression analysis showed that high expression levels predicted poor survival.2.2.2 Transient and stable transfection of NAP1L1 was performed in breast cancer cell lines,and MTT,EDU and plate cloning were used to confirm that NAP1L1 promoted the proliferation of breast cancer cells.One of the stable NAP1L1 breast cancer cell lines was used for subcutaneous tumor formation in nude mice,further confirming that NAP1L1 is an oncogene.2.3 NAP1L1 interacts with HDGF to recruit c-jun and stimulate CCND1 to promote the proliferation of breast cancer cells2.3.1 Interaction between NAP1L1 and HDGF:binding of NAP1L1 and HDGF was confirmed by endogenous Co-IP experiments.Confocal microscopy showed that NAP1L1 and HDGF co-localized in the cytoplasm of breast cancer cells.2.3.2 HDGF recruitment of c-Jun:Co-IP analysis showed an interaction between c-Jun and HDGF in breast cancer.2.3.3 HDGF transfection increased c-Jun/CCND1 signal and restored NAP1L1 to inhibit the proliferation of breast cancer cells:qRT-PCR and Western blot analysis confirmed that HDGF expression was up-regulated.In vitro,cell proliferation and EDU staining were significantly restored.The signal intensity of c-Jun/CCND1 was significantly enhanced by Western blot.2.3.4 C-Jun transfection enhanced CCND1 signal in breast cancer cells and restored proliferation of NAP1L1 inhibited cells:C-Jun-cDNA plasmid transfected NAP1L1 suppressor cells and restored the ability of NAP1L1 suppressor cell proliferation and EDU staining in vitro.3.Conclusion:3.1 NAP1L1 protein expression was significantly increased in breast cancer tissues,Its elevated expression was an unfavorable factor for breast cancer clinical progression and poor prognosis.3.2 NAP1L1 promotes the proliferation of breast cancer cells.3.3 NAP1L1 interacted with the hepatoma-derived growth factor(HDGF),and the latter subsequently recruited the key oncogenic transcription factor C-JUN,which finally induced the expression of cell cycle promoter CCND1 and thus induce the cell growth of breast cancer.