Function And Mechanism Of Action Of CBX8 In The Progression Of Hepatocellular Carcinoma

Background and objectivePrimary liver cancer(PLC)is a common malignancy worldwide.According to the 2018 global cancer statistics,it has the sixth highest global incidence and fourth highest death rate.PLC mainly includes hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC),and the HCC-ICC combination,among which HCC accounts for approximately 85-90%of total number of cases.Therefore,PLC,in this article,is hereafter referred to as HCC.Despite advancements in surgical resection,liver trans-plantation,molecular-targeted therapy,and immunotherapy,the poor prognosis of HCC has still persisted over the last decades.HCC is typically diagnosed at an advanced stage and has been associated with a high recurrence rate,resulting in adverse outcomes in patients.Therefore,it is critical to better understand the molecular mechanisms underlying HCC progression and identify novel therapeutic targets for the treatment of patients with HCC.Among the pathways and factors involved in the development and maintenance of HCC,polycomb-group(PcG)proteins have drawn the most attention of researchers.Deregulation and dysfunction of PcG proteins often lead to the blockade or inappropriate activation of developmental pathways,leading to the enhancement of cancer cell proliferation,inhibition of cancer cell apoptosis,and increase in the cancer stem cell population.Chromobox homolog 8(CBX8)is an important member of the PcG protein complex.Recently,it was reported that CBX8 is abnormally expressed in multiple tumor types and plays a critical role in promoting the progression of malignant tumors.However,the mechanisms by which CBX8 regulates the malignant progression of HCC remain unclear.Therefore,in this study we aimed to investigate the role of CBX8 in HCC progression and its mechanism of action.Methods1.Investigation of the correlation between CBX8 and cell cycle signaling pathwaysGene set enrichment analysis(GSEA),qRT-PCR,and western blot analysis were used to investigate the relationship between CBX8 and cell cycle signaling pathways.2.Exploration of the mechanism of the CBX8-mediated cell cycle regulationBioinformatic tools and co-immunoprecipitation assay were used to identify and verify the proteins that bind to CBX8.After over-expressing CBX8 and then interfering with YBX1,qRT-PCR and western blot analysis were conducted to elucidate the mechanism through which CBX8 regulates the cell cycle.3.Exploration of the biological function of CBX8The EdU and CCK-8 assays were performed to assess the effect of CBX8 on the proliferation of HCC cells.4.Exploration of the mechanism through which CBX8 regulates the proliferation of HCC cellsThe EdU and CCK-8 assays were performed to elucidate the mechanism of CBX8 in regulating the proliferation of HCC cells.5.Exploration of the expression and clinical significance of CBX8 in HCCqRT-PCR and western blot analysis combined with TCGA database analysis were conducted to assess the expression of CBX8 in HCC patients and its relationship with HCC prognosis.Results1.The expression of CBX8 is positively correlated with that of cell cycle signaling pathway membersThrough gene enrichment analysis of the HCC TCGA database,the expression of CBX8 in HCC was found to be positively correlated to the cell cycle signaling pathway.After specifically interfering with CBX8 expression,qRT-PCR screening of cell cycle genes revealed that CBX8 mainly regulates cell cycle progression through CyclinD1.qRT-PCR and western blot analysis revealed that CBX8 can regulate CyclinD1 at both gene and protein expression levels.2.CBX8 regulates the expression of CyclinDl through YBX1Using bioinformatics tools and co-immunoprecipitation analysis,we confirmed that CBX8 interacts with YBX1.Using qRT-PCR and western blot analysis,we found that CBX8 can regulate the expression of CyclinD1 at both mRNA and protein expression levels via YBX1.3.CBX8 regulates HCC cell proliferationUsing EdU and CCK-8 assays and nude mouse subcutaneous tumor experiments,we found that CBX8 promotes the proliferation of HCC cells.4.CBX8 regulates HCC cell proliferation via YBX1Using EdU and CCK-8 assays,we found that CBX8 can promote the proliferation of HCC cells via YBX1.5.CBX8 is highly expressed in HCC and is associated with poor prognosisUsing qRT-PCR and western blot analysis combined with TCGA and GEO database analyses,we confirmed that CBX8 is highly expressed in HCC cells.Patients with high CBX8 expression were found to exhibit reduced overall survival and disease-free survival.HCC patients with concurrent high expression levels of CBX8 and YBX1 also exhibited reduced overall survival and poor prognosis.Conclusions1.CBX8 expression is positively correlated with cell cycle signaling pathway2.CBX8 increases the levels of CyclinD1 via YBX13.CBX8 promotes HCC cell proliferation4.CBX8 promotes HCC cell proliferation via YBX15.CBX8 expression is up-regulated in HCC cells,and its expression is correlated with patient prognosis.