The Mechanism Of HMGB1 Interacting With BRG1 To Promote The Proliferation And Metastasis Of Prostate Cancer

Background and purposeAs a chromatin-binding protein,HMGB1 plays an important role in the development of many tumors.Our previous studies have confirmed that HMGB1 and its receptor RAGE are highly expressed in prostate cancer tissues,and their expression in prostate cancer tissues is correlated with tumor invasion depth,local lymph node metastasis and distant metastasis;abnormally expression of HMGB1 and co-expression of it and its receptor suggest a poor prognosis in patients with advanced prostate cancer.Hence,our group intends to further study the expression and function of HMGB1 in prostate cancer cells,in order to elucidate its molecular regulation mechanism in the proliferation and metastasis of prostate cancer.MethodsReal-time quantitative PCR and Western blot were used to detect the expression of HMGB1 in human prostate cancer cell lines and prostate epithelial cell lines.The expression of HMGB1 was determined by utilizing immunohistochemical technique in 64 human prostate cancer tissue microarrays with follow-up data.The correlation between the expression level of HMGB1 and the clinicopathological parameters of patients with prostate cancer were analyzed.The stable of knockdown or overexpression HMGB 1 and control prostate cancer cell lines were established by using the lentiviral vector.After silencing or up-regulation of HMGB 1 the proliferation change of prostate cancer cells in vitro was measured by using CCK-8,EdU staining and cloning formation assays.Flow cytometry was used to analyze the changes of cell cycle distribution.Scratch healing experiments and Transwell chamber migration as well as Matrigel invasion assays were used to detect the migration and invasion ability of HMGB1 on tumor cells in vitro.Nude mice subcutaneous tumor formation test,tail vein injection tumor formation experiments were utilized to observe the tumor growth and lung metastasis ability of prostate cancer cells.The activation of PI3K/Akt pathway was evaluated by utilizing Western blot after HMGB1 expression was altered.The binding proteins of HMGB1 were screened by IP-binding combined with mass spectrometry.The interaction between HMGB1 and BRG1 was further confirmed by immunofluorescence confocal and co-iimunoprecipitation(Co-IP)experiments.Changes in key molecular markers protein levels of epithelial-mesenchymal transition(EMT)was detected in silencing of BRG1 with stably overexpressing HMGB1 cells or ectopic of BRG1 with silencing HMGB1 cells.To confirm the critical role of BRG1 in regulation of the downstream signaling pathways by HMGB1.The recovery experiment further confirmed that BRG1 was involved in HMGB1-mediated carcinogenic effects.Correlation analysis of protein expression levels of HMGB1 and BRG1 in 64 prostate cancer samples was performed using Spearman correlation method.It is preliminary revealed that HMGB1 is involved in the possible molecular mechanism of prostate cancer proliferation and metastasis.ResultsThe mRNA expression level of HMGB1 in human prostatic cancer cell lines was significantly higher than that of immortalized normal epithelial cells,and the abnormally expressed HMGB1 was positively correlated with Gleason score and clinical stage of prostate cancer patients.Overexpression of HMGB1 in prostate cancer cells facilitated cell proliferation,migration and invasion in vitro accompanied with acceleration of tumor formation and lung diffusion and metastasis ability.The results were reversed in prostate cancer cells with HMGB1 interference.Co-IP and immunofluorescence experiments confirmed the interaction between HMGB1 andBRG1.BRG1 expression was up-regulated in cells with overexpression of HMGB1,which may promote the EMT of cells by stabilizing the transcription factor ZEB1 and inhibiting the tumor epithelial marker E-cadherin.Silencing of BRG1 in HMGB1 overexpressing cell lines effectively attenuated activation of PI3K/Akt signaling pathway that was induced by HMGB1 and its mediated carcinogenic effects were also alleviated.The results were opposite in upregulation of BRG1 with silencing HMGB1.Theose indicated that the stability of BRG1 is critical for the activation of PI3K/Akt signaling pathway induced by HMGB1.In 64 prostate cancer tissues,the protein expression level of HMGB1 was positively correlated with the expression of BRG1 protein,in addition the expression of BRG1 was positively correlated with Gleason grade and clinical stage of prostate cancer patients.ConclusionThe expression of HMGB1 is elevated in prostate cancer cell lines and cancer tissues,and its expression level is closely related to the malignant progression of prostate cancer.By binding to the BRG1 protein,HMGB1 is involved in the activation of the PI3K/Akt signaling pathway and thus promotes the EMT process of prostate cancer,ultimately leading to tumor proliferation and metastasis.In conclusion,this study reveals that the HMGB1-BRG1 axis can serve as a potential target for the regulation of prostate cancer growth and metastasis.