BRD9 Promotes Hepatocellular Carcinoma Progression Via Activating The Wnt/β-catenin Signaling Pathway

Background: Hepatocellular carcinoma(HCC)is one of the leading causes of cancer deaths worldwide.It is difficult to diagnose in the early stage,and when it develops to the advanced stage,treatment options are limited and the treatment effect is not ideal.Because it is easy to metastasize and has a high recurrence rate,patients with HCC have a poor prognosis.Clarifying the mechanism of the occurrence and development of HCC will help to discover biomarkers and develop effective drugs for HCC.Epigenetic regulation dysfunction plays a significant role in the occurrence and development of tumors.Among them,BRD9-mediated histone acetylation modification is an important way of epigenetic regulation.BRD9 contains a bromodomain,which is a subunit of the SWI-SNF nucleosome remodeling complex.The bromodomain can specifically recognize the acetylated lysine residues on the histone tail,thereby regulating the assembly of the chromatin complex and the transcription of downstream gene.Recent studies have shown that BRD9 plays an important role in several tumors,including acute myeloid leukemia,synovial sarcoma,squamous cell lung cancer and HCC.However,the biological function and molecular mechanism of BRD9 in HCC are still unclear,and further research is necessary.Methods: The BRD9 mRNA and protein expression in HCC tissues,normal liver tissues,HCC cell lines,and normal liver cell lines were detected by database analysis of the cancer genome atlas(TCGA),RT-PCR assays,and western blot assays.Logistic regression analysis and Wilcoxon rank sum test were used to evaluate the correlation between BRD9 expression and clinicopathological characteristics in patients with HCC in the TCGA database.Kaplan-Meier survival analysis and Cox regression analysis were used to study the relationship between BRD9 expression and prognosis.GSEA software was used to perform GO function analysis and KEGG signaling pathway analysis to predict the biological functions and signaling pathways that may be affected by BRD9.Lentiviruses carrying p Lenti-CMV-Puro-BRD9 and p LKO.1-BRD9-sh RNA were used to up-regulate or knockdown the expression of BRD9 in Huh7 and MHCC-97 H HCC cell lines,respectively.In vitro,CCK-8,clone formation,Ed U proliferation assays and flow cytometry were used to observe the effects of BRD9 on the proliferation,cell cycle and apoptosis of HCC cells.An HCC xenograft tumor model was established to observe the effect of BRD9 on tumor growth in vivo.In order to clarify the mechanism of BRD9 in HCC,western blot assays and rescue assays were performed to confirm that BRD9 affected the malignant phenotype of HCC through the Wnt/β-catenin signaling pathway.Results: Compared with normal liver tissues and hepatocytes,BRD9 m RNA and protein expression in HCC tissues and HCC cell lines were increased.BRD9 expression level was positively correlated with Grade,Stage,T classification and poor prognosis of HCC.BRD9 could be used as an independent risk factor for the overall survival of patients with HCC.GO enrichment analysis found that in samples with high expression of BRD9,the gene sets that regulate cell proliferation,cell cycle and cell apoptosis were significantly enriched.In vitro,up-regulating BRD9 could promote the proliferation and cell cycle process of Huh7 cell line and inhibit its apoptosis,while down-regulation of BRD9 could inhibit the proliferation and cell cycle process of MHCC-97 H cell line and promote its apoptosis.In vivo,BRD9 overexpression could significantly promote the growth of HCC xenograft tumor in nude mice,while BRD9 knockdown could significantly inhibit tumor growth.KEGG enrichment analysis found that in samples with high expression of BRD9,the gene set that regulates the Wnt signaling pathway was significantly enriched.Rescue assays proved that the Wnt signaling pathway could be activated by BRD9,and inhibition of this pathway could reduce the cancerpromoting effect of BRD9.Conclusion: BRD9 is significantly up-regulated in HCC and is not conducive to the survival and prognosis of patients with HCC.BRD9 may promote the development of HCC by activating the Wnt/β-catenin signaling pathway.Therefore,BRD9 may be a potential biomarker and therapeutic target for patients with HCC.